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Isolation and biophysical studies of horse plasma gelsolin Ruiz Silva, Beatriz Eugenia 1991

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I S O L A T I O N AND B I O P H Y S I C A L HORSE  PLASMA  STUDIES  OF  GELSOLIN  By Beatriz B.Sc,  Eugenia  Universidad  A THESIS  IN  REQUIREMENTS DOCTOR  Silva  Michoacana,  SUBMITTED  THE  Ruiz  OF  Mexico,  1982  PARTIAL  FULFILLMENT  FOR  DEGREE  THE  OF  PHILOSOPHY  in  THE  FACULTY  OF  (DEPARTMENT  We  accept to  THE  this  the  OF  thesis  April  OF  STUDIES  CHEMISTRY)  required  UNIVERSITY  ©Beatriz  GRADUATE  as  conforming  standard  BRITISH  COLUMBIA  1991  Eugenia  Ruiz  Silva,  1991  OF  In  presenting this  degree at the  thesis  in  partial  fulfilment  of  the  requirements  University  of  British Columbia, I agree that the  for  an  advanced  Library shall make it  freely available for reference and study. I further agree that permission for extensive copying  of  department  this thesis for scholarly or  by  his  or  her  purposes may be granted by the  representatives.  It  is  understood  head of my  that  publication of this thesis for financial gain shall not be allowed without permission.  Department The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ^ T  R  , - J  IT,  /?<?/  copying  or  my written  ABSTRACT  Gelsolin and of  from  reproducible 1.4  to  ml/(mg  other  an  kDa.  chain  temperature calcium The  «  1.5  M  the  actin  and  54  is  of  of  amino  composition  in  acid  migrates  as  gel 90  is  a  kDa.  the  the  globular  protein  profiles i t s  transition  in of  gelsolin  were  melting  the  absence  the  in  of  i n t r i n s i c  The  4 6 °C  presence  of  single  Hydrodynamic  values. Tm «  a  electrophoresis  measuring  unfolding  able  to  filaments. of  Gelsolin  presence  °C  good  coefficient  of  were  in  absorption  denaturation  by  obtained  isolated  the was  divalent guanidine  found  to  be  denaturant.  solutions  (acrylodan)  mass  chemical  mid-point  polymerization  fluorescent  It  e l l i p t i c i t y  Tm «  been  in  gelsolin  g e l s o l i n  values  abolition  actin  an  has  similar  that  and  Gelsolin sever  It  molecular  hydrochloride-induced at  has  polyacrylamide  and  for  fluorescence  cation.  in  suggest  Thermal  is  plasma  g e l s o l i n s .  apparent  obtained  of  and  plasma  calculations 75  yields.  cm)  polypeptide with  horse  the  and  These  lag  the  phase  polymerized  probe  in  in  are  time  steady  the  with  the  polymerization  actin  manifested course  state  presence  and  of  of  by  actin  viscosity  of  gelsolin.  labelled  with  the  6-acryloyl-2-dimethylaminonaphthalene  produce  calcium.  actin  activities  decreased  interacts  to  nucleate  a  Upon  2:1  actin:gelsolin  chelation  ii  of  the  complex divalent  in  the  cation  from  t h e 2:1 c o m p l e x ,  one a c t i n  molecule  i s released  p r o d u c i n g a 1:1 E G T A - r e s i s t a n t c o m p l e x . The  fluorescence o f 2-(N-methylanilino)naphthalene-6-  sulphonate  (MANS), a f l u o r e s c e n t p r o b e t h a t i s s e n s i t i v e t o  the p o l a r i t y o f i t s environment, shifted  upon  binding  i s b o t h enhanced and b l u e  to gelsolin.  These  results are  i n d i c a t i v e o f t h e b i n d i n g o f MANS t o h y d r o p h o b i c g e l s o l i n . G e l s o l i n b i n d s 2.5 ± 0.9 m o l e c u l e s  regions i n  o f MANS w i t h a  d i s s o c i a t i o n c o n s t a n t o f 0.24 ± 0.13 |1M. Gelsolin cations  c a n be l a b e l l e d  with  the  pyrenyl)iodoacetamide integrity.  i n t h e absence o f d i v a l e n t  sulfhydryl-specific  probe  N-(l-  (PIA) w i t h o u t a l t e r i n g i t s s t r u c t u r a l  The l a b e l l e d  protein  presents  excimer-like  pyrene emission i n d i c a t i v e o f the p r o x i m i t y o f t h e l a b e l l e d cysteines  i n the three  dimensional  structure  of the  protein.  The s p e c t r o s c o p i c c h a r a c t e r i s t i c s o f t h e l a b e l l e d  protein  indicate  that  t h e excimer  emission  arises  from  i n t e r a c t i o n s t h a t c a n be t r a c e d t o t h e g r o u n d s t a t e o f t h e pyrene molecules. excimer  This  i s i n contrast t o the well  e m i s s i o n t h a t a r i s e s from molecules  o t h e r i n t h e ground s t a t e .  iii  studied  t h a t r e p e l each  TABLE OF CONTENTS  page ABSTRACT  i  L I S T OF TABLES  i viii  L I S T OF FIGURES  ix  ACKNOWLEDGMENTS  x i i  ABBREVIATIONS  xiii  INTRODUCTION  1  A. G e l s o l i n : An A c t i n - B i n d i n g P r o t e i n  1  B. E x c i t e d S t a t e D e a c t i v a t i o n P a t h w a y s . An o v e r v i e w .  8  B.l.  Fluorescence P o l a r i z a t i o n  13  B.2. E x c i t e d S t a t e Q u e n c h i n g  16  B.3. E x c i m e r F o r m a t i o n  17  B. 4. E x c i t a t i o n E n e r g y T r a n s f e r  20  C. L u m i n e s c e n c e  Probes i n t h e Study o f P r o t e i n s  21  C. l . I n t r i n s i c P r o b e s  22  C.2. E x t r i n s i c P r o b e s  23  C.2.a. N o n c o v a l e n t P r o b e s t o S t u d y P r o t e i n s . . . C.2.b. M o l e c u l e s t h a t B i n d to  23  Covalently  Proteins  C.2.c. M e t a l I o n s  RESULTS AND DISCUSSION  25 25  27  PART I . I s o l a t i o n o f G e l s o l i n f r o m H o r s e P l a s m a  27  PART I I . P h y s i c o c h e m i c a l C h a r a c t e r i z a t i o n  32  A.  Absorption Coefficient  32  B.  Molecular  33  C.  Amino Acid. C o m p o s i t i o n  34  D.  Sedimentation  36  E.  Molecular  Weight  by  Coefficient  Weight  Determined  SDS-PAGE  by  and  Gel  F.  UV A b s o r p t i o n a n d  G.  Circular  H.  Stability  Stokes'  Radius  Filtration  38  Florescence  Emission  Spectra...  Dichroism Spectra to  Temperature  44  and  Chemical  Denaturants PART  III.  Difference  B.  Viscometry  C.  Interaction with  46  Interaction  A.  with  Actin  54  Absorbance  55 57  of  Gelsolin  with  Actin  Labelled  Acrylodan  58  C.l.  Spectral  Characteristics  C.2.  Calcium Sensitivity  and  59 Stoichiometry  of  Binding D.  Circular of  PART  IV.  62  D i c h r o i s m and  Gelsolin  and  its  Interaction  of  with  the  of  UV A b s o r p t i o n  Binding Horse  Hydrophobic  A.  Binding  B.  Dissociation Constant  C.  Effect  D.  Thermal  E.  Correlation  of  42  MANS  MANS  to  on  Actin  Plasma  67  Gelsolin  Fluorescent  Probe  MANS..  Gelsolin and Number  of  of  of  v  Binding and  MANS-Gelsolin  MANS-Gelsolin  70 71  Gelsolin Activity  Denaturation Time  to  Studies  Sites.  71  Structure.  74  '..  75 77  PART V .  Covalent  Labelling of  Fluorescent A Probe  of  Probe  Labelling of  B.  Degree  C.  Emission  D.  Ground State  F.  Lifetime  Measurements  G.  Activity  and  of  Proximity  Gelsolin with  Labelling of  PIA  PIA-Gelsolin  Characteristics  87 88  Interactions  91 103  Structure  of  PIA-Gelsolin  104  109  EXPERIMENTAL  SECTION  Preparation  A.l  81  89  CONCLUSIONS  A.  The  N-(1-pyrenyl)iodoacetamide.  Cysteines  A.  Gelsolin with  Gelsolin  of  113  proteins  113  Isolation  113  A . l . a .  Preparation  Plasma  113  A.l.b.  Gelsolin Purification  113  A.2.  Actin  of  Isolation  114  B.  SDS-PAGE  115  C.  Amino A c i d A n a l y s i s  116  D.  Sedimentation  116  E.  Gel  F.  Viscometry  G.  Labelling  Filtration  117 118  of  Gelsolin with  Degree  of  Labelling of  Labelling  of  Actin with  of  Labelling of  G. l . H.  Velocity  H . I.  Degree  vi  PIA PIA-Gelsolin  Acrylodan ADA-actin  118 119 119 120  I.  Absorption Spectroscopy  J.  Fluorescence  Spectroscopy  ..  120 121  J . l .  Steady  State  Measurements  121  J.2.  Fluorescence  Polarization  121  J.3.  Lifetime  K.  Circular  L.  Nuclear  Measurements  D i c h r o i s m Measurements Magnetic  Resonance  REFERENCES  122 122 123  124  vii  LIST  Table I.  OF  TABLES  Caption Calculation  of  the  Page  Absorption  Coefficient  of  Gelsolin II.  Amino  III.  Summary of  IV.  V.  VI.  Acid  the  of  and  the  on  Effect  Gelsolins  of  MANS  Gelsolin  p  Values  Parameters  on  on  G-Actin of  Polymerization  Actin  Activity  Filaments  and  Structure  the  Excitation  PIA-DTT,  and  PIA-ME,  Absorption  PIA-Gelsolin  PIA-Actin  Proton  NMR  94  Spectral  Data  of  PIA  in  DMF  DMF/buffer  100  Thermally-Induced  Activity  of of  58  75  for  of  35  53  Viscosity  Parameters IX.  Plasma  Physicochemical  Gelsolin  of  and  of  Gelsolin  Effect  and  VIII  Composition  of  Spectra  VII  31  Changes  on  PIA-Gelsolin  PIA-Gelsolin  viii  at  the 483  on A c t i n  Decay nm  101  Polymerization.  105  LIST  Figure Polymerization  2.  Actin  3.  Jablonski  4.  Emission in  Binding  Actin  3  Proteins  5 9  Characteristics  of  Pyrene  Solutions  Cyclohexane  Study  18  Diagram  of  Luminescent  Probes  Proteins  6.  Isolation  of  7.  Molecular  Mass  8.  Typical  9.  Intrinsic  10.  Determination of  of  Page  Diagram  Schematic to  11.  FIGURES  Caption  1.  5.  OF  Gelsolin of  Schlieren  Horse  from Horse  Gelsolin  of  Determination by  of  Gel  the  30  SDS-PAGE  34 36  Coefficient  Molecular  G e l s o l i n by Stokes'  of  Gelsolin.  Absorption Spectrum  13.  Polypetide  14.  Far  15.  Thermal  of  Reference  Gel  Radius  Filtration of  Horse  Plasma  Gelsolin  43  Data  44  of  Gelsolin  as  of  Gelsolin..  Followed  Fluorescence 16.  Thermal  Denaturation  Unfolding of  45  by 47  of  Gelsolin  Dichroism 17.  39  40  Dichroism Spectrum  Denaturation  37  Mass  Filtration  12.  UV C i r c u l a r  by  Plasma  Pattern  Sedimentation  Plasma  Gelsolin  22  by  Circular 4 9  Gelsolin  by  ix  Gu-HCl  50  18.  Schematic  Representation of the Effects  o f G e l s o l i n on A c t i n P o l y m e r i z a t i o n 19.  N u c l e a t i o n A c t i v i t y o f Horse Plasma G e l s o l i n on A c t i n P o l y m e r i z a t i o n  20.  55  Schematic  Representation o f the  56 Interaction  of A c t i n and G e l s o l i n  58  21.  Fluorescence Emission Spectra o f ADA-actin  60  22.  Stern-Volmer  P l o t s f o r the Quenching o f A c r y l o d a n  i n A c t i n and i n t h e A c r y l o d a n - L a b e l l e d A c t i n - G e l s o l i n Complex  61  23.  T i t r a t i o n o f ADA-Actin  with Gelsolin  24.  P o l a r i z a t i o n Increase i n the Fluorescence o f G e l s o l i n upon B i n d i n g t o A c t i n  25.  with Actin  69  E m i s s i o n S p e c t r a o f MANS i n B u f f e r , G e l s o l i n and  27.  66  C o n f o r m a t i o n a l Changes i n G e l s o l i n upon Interaction  26.  63  Isopropanol  72  T i t r a t i o n s o f the Increase i n the Intensity  Fluorescence  o f MANS a t 420 nm  73  28.  Thermal D e n a t u r a t i o n o f MANS:Gelsolin  76  29.  P e r r i n P l o t f o r MANS : G e l s o l i n  78  30.  P o t e n t i a l Energy Diagrams f o r t h e Ground and E x c i t e d S t a t e s o f M and Q  31.  83  H y p o t h e t i c a l P o t e n t i a l Energy Curves o f Adsorbed Pyrene  85  32.  E x c i t a t i o n and Emission S p e c t r a o f P I A - G e l s o l i n . . .  90  33.  M o d e l Compounds PIA-DTT a n d PIA-ME  92  X  34.  PIA-Gelsolin with  35.  Excimer  Excitation  Proposed Model Characteristics  36.  Quenching the  and Decay  Curves  together  Pulse to of  Studies  96  Explain  the  Spectroscopic  PIA-Gelsolin of  the  Decay  of  PIA-Gelsolin  103  Stability  of  PIA-Gelsolin  104  Excimer  37.  Thermal  38.  Temperature  Effect  on t h e  PIA-Gelsolin  Two  Components  97  Fluorescence  of  of 106  xi  ACKNOWLED GEMENT S I thank Professor L.D. Burtnick for his guidance during the preparation of this t h e s i s . I would also l i k e t o t h a n k Ms D a n a Z e n d r o w s k y , an I n s t r u c t o r w i t h c o n t a g i o u s e n t h u s i a s m , f o r making T A ' i n g an invaluable experience for a l l the fortunate TA's of Chem 2 3 0 . A l s o , . f o r h e r g r e a t human q u a l i t y . I w i l l a l w a y s be i n debt t o P r o f e s s o r N.J. Turro from Columbia University for his h o s p i t a l i t y and continuous advice during the w r i t i n g of t h i s t h e s i s . Special thanks f o r h i s guidance and s u p e r v i s i o n during the p r e p a r a t i o n of PART V o f t h i s t h e s i s . The "Turro group" is a place of c o n t i n u o u s l e a r n i n g . Thanks a r e due t o Dr K.R. Gopidas for sharing his expertise i n measuring the lifetimes presented in the thesis. Thanks to Miguel, who b e s i d e s being my inspiration and example has given time for advice and e n c o u r a g e m e n t , m a k i n g my w o r k easier. Financial support from the University of B r i t i s h Columbia in the form of a University Graduate Fellowship and f r o m C o n s e j o N a c i o n a l de C i e n c i a y T e c n o l o g i a (Mexico) to finish this thesis, is also greatly appreciated. T o c l o s e w i t h a g o l d e n s e a l : q u i e r o a g r a d e c e r a mama y a papa por el ejemplo y motivacion al e s t u d i o que nos i n c u l c a r o n desde pequefias. A mis Ninas Conchita y Josef ina "mis queridas maestras". A mis hermanos y a la familia Mendoza por l a s p o r r a s . G r a c i a s a I n g r i d por escuchar las p l a t i c a s que a l g u n a v e z t u v e que p r e s e n t a r y p o r su v a l i o s a e i n u m e r a b l e ayuda en e l l a b o r a t o r i o . G r a c i a s a M i g u e l p o r e l a m o r e i n s p i r a c i o n q u e me d a y p o r q u e j u n t o s v e m o s q u e e s t a t e s i s no e s e l f i n a l s i n o un p a s o en l a r e a l i z a c i o n de nuestros planes.  xii  Abbreviations Acrylodan  ADA  (6-acryloy1-2-dimethylamino-  naphthalene) ADA-Actin  Actin  ATP  Adenosine  .  Buffer  A  labelled  with  triphosphate  2 mM T r i s - H C l , 0.2  acrylodan  mM A T P ,  1 mM D T T ,  pH -  0.2  mM  CaCl2,  7.6  Buffer  D  2 0 mM M O P S ,  1 5 0 mM K C 1 ,  1 mM E D T A ,  pH =  7.6  Buffer  E  2 0 mM M O P S ,  1 5 0 mM K C 1 ,  1 mM E G T A ,  pH =  7.2  CD  Circular  DTT  Dithiothreitol  EDTA  Ethylenediaminetetraacetic  EGTA  dichroism acid  Ethylenebis(oxyethylenenitrilo)tetraacetic acid  G-Actin  Globular  Gu-HCl  Guanidine  F-Actin  Filamentous  MANS  actin hydrochloride actin  2-(N-Methylanilino)naphthalene-6-sulfonate  MOPS  4-(N-Morpholino)propanesulphonic  ME  Mercaptoethanol  PMSF PIA PIA-Gelsolin PM SDS-PAGE  Phenylmethylsulphonyl  fluoride  N-(1-Pyrenyl)iodoacetamide Gelsolin  labelled  with  PIA  N-(1-Pyrenyl)maleimide Sodium  dodecylsulphate-polyacrylamide  electrophoresis Tris  acid  T r i s (hydroifymethyl)  xiii  aminomethane  gel  INTRODUCTION  A. G e l s o l i n . An A c t i n - B i n d i n g  Gelsolin was  named  Protein.  i s an abundant p r o t e i n after  transformations  i t s ability  i n cytoplasmic  i n vertebrates  to  perform  extracts.  gel-sol  Gelsolin  1  exists  b o t h a s an i n t r a c e l l u l a r a n d a s a s e c r e t e d p r o t e i n . forms  have  similar  characteristics, extension as  functional  differing  only  that  and  in a  Both  2  structural  25  amino  acid  a t t h e amino t e r m i n u s o f t h e e x t r a c e l l u l a r  compared t o t h e i n t r a c e l l u l a r p r o t e i n . ' 3  4  form  The b i o l o g i c a l  i m p o r t a n c e o f g e l s o l i n r e l i e s on t h e f a c t t h a t  i t binds t o  actin. Actin and  i s one o f t h e most  i t can e x i s t  globular  actin)  as a s i n g l e o r as  d e p e n d i n g on f a c t o r s  a  abundant p r o t e i n s molecular  filamentous  unit  i n nature (G-actin  polymer  (F-actin)  s u c h a s pH, t e m p e r a t u r e , t h e p r e s e n c e  of s a l t s and t h e presence o f a c t i n - b i n d i n g  proteins.  The p o l y m e r i z a t i o n o f a c t i n h a s b e e n s t u d i e d detail.  In t h e absence o f o t h e r  proteins steps: ' 8  or  i t has been  shown  binding  to consist  or  5 - 1 0  i n some  interacting  of at  least  1 1  (a) Monomer a c t i v a t i o n . T h i s i s a c o n f o r m a t i o n a l change i n t h e s t r u c t u r e  of a c t i n that  results i n a  4  form is  that  known  than (b) of  is to  that  stable  (c)  stage  nucleus  filament ends  (B  nucleus  the  (d)  P  of  polymer  and  lengthening manifested  viscosity  a  may  is  decrease  (Figure by  induced la) .  taking  by  This  process  advantage  differences  between  of  monomers  it  slow  the  actin  cessation also  actin  salts be  actin  possible  growing  viscosity  after  can  end  such  that  monomer  A of  for  events of  are  polymeric  recovery  of  the  be  deduced  polymerization.  followed  -2-  The  filaments.  sonication.  can  with  la).  upon  of  is  These  in  the this  F-actin.  properties  the  a  polymerization  addition  At  mechanisms  processes  the  growth.  to  possible  modeling of  Experimentally, easily  are  grow.  monomers  and  and  of  actin  dissociation.  figure  annealing  occur  of  formation  three can  actin  different  in  observed  kinetic  rate  shortening  These  of  growing  annealing  as  sonication. through  are  chain  filament  the  fast  and  and  which  form  different  the  about  actin  association  exceeds  Breaking  actin  in  respectively  Breaking  This  spectrum  requires  polymer  Addition of  distinguish a and  the  results  rate ends  of  UV a b s o r p t i o n  consisting of  from which  the  proteolysis.  Polymerization  Elongation.  stable  a  to  G-actin.  Nucleation. a  resistant  exhibit  of  molecules  to  more  of  actin  as  MgCl2  in  and  several  show and  can  be KC1 ways  s i g n i f i c a n t  polymer.  Among  these flow  properties  we  find:  birefringence  and  iodoacetamide-labelled salt lb)  induced is  t h i r d  time  found to power  depends accounts  of  for  attributed  to  course  actin  the  of  of  a  By  any  actin slow  on  actin slow  elongation  of  absorbance,  of  these  p y r e n y l methods,  polymerization  phase  concentration  i n i t i a l  the  difference  f l u o r e s c e n c e  actin.  consist  d i r e c t l y  viscosity,  that  and  a  of  and  the  the  on  phase  concentration. phase,  (Figure  depends  fast  the  the that  Nucleation  rapid  phase  is  filaments.  J  T  1-  Time  (tain)  Figure 1. P o l y m e r i z a t i o n o f A c t i n . A sample of monomeric a c t i n i s induced t o polymerize by adding s a l t at time equal zero, (a) Schematic representation of a c t i n assembly, (b) Time course of the polymerization of actin induced by s a l t s . The p l o t i s c o n s t r u c t e d w i t h t h e changes o b s e r v e d in the measured property against t i m e . 8  C o n d i t i o n s polymerization. actin  is  i n In  l i v i n g  muscle  contractile  c e l l s  cells,  activity,  where most  -3-  of  s h o u l d  the the  main  favour  function  actin  is  of  found  in  polymeric  more by  dynamic  several  binding and  state  regulated  have  to  Sequestering  actin  polymerize.  In  monomers  associate  filament.  By  the  net  regulate  the  accelerating actin  more  effect  Capping  (2)  of  trapping of and  polymerization  (slow)  the  proteins  may  severing  previously  from the  also  polymer  (3) polymeric  This  reduce formed  their  the  proteins them  the  to  actin  ends  of  sequestering  the  proteins  F-actin.  step  These  in  proteins  filaments the  time  results faster  in than  polymer  filaments  at  by  course  shorter, during  capping protein.  average  actin  on  6  allow  actin  the  sources  are  the  a  Actin  effects  These  at  in  manner  different  polymer,  form  of  precise  not  effect  that  absence  These  length sites  by away  ends.  Crosslinking actin  of  found  proteins.  Proteins.  nucleation  filaments in  monomers,  length  polymerization.  abundant  formed  Severing  is  categories:  do  depolymerizing  average  the  and  dissociate  actin  to  Proteins.  already and  from  major  monomers  an  binding  according three  actin  remarkably  isolated  c l a s s i f i e d  Monomer  a  actin  been  into  cells,  in  called  polymerization  bind  have  non-muscle  proteins  been  (1) that  In  proteins  have  actin  form.  to  Proteins.  produce  These  networks  filaments.  -4-  proteins or  bundles  crosslink of  actin  Different  actin  binding  combination  of these  main  non-muscle  c e l l s  dimensional  structures  i s  activities.  found  MOltOMER SEQUESTRATION  proteins  i n  (Figure  a  may show  As a result,  wide  variety  one o r actin of  a i n  three  2).  CAPWMG  CROSSUHKItlO  Short Capped Filaments  * » Moncraar Bound to DepolynBrzlng Protein Monomers  Long Filanent  Network Bundle  Figure 2. Actin Binding Proteins. Monomeric a c t i n (.), actin p o l y m e r ( T r > ). The a c t i n binding proteins are represented by the following symbols: capping and severing proteins ([), monomer b i n d i n g p r o t e i n s ( c ) , crosslinking proteins(~). 6  The actin and  dynamic  plays  c e l l  an  These  have  important  shape.  polymerization been  interconversion  In  this  through shown  role  of  the various  i n  governing  respect,  gelsolin  multiple t o occur  and complex i n at  ways:  -5-  least  c e l l  forms  of  motility  affects  actin  interactions. three  1  2  different  (1)  Severing  (2)  Capping  (3)  Nucleation It  of  is  also  is  one  result the  been  the  the  of  cell  filaments  clot  micromolar  its both  conditions  2  severs  effect  on  3  c a p i l l a r i e s  binding  2  0  -  In  2  2  plasma  viscosity. actin  the  As  way  a  into  potential interfere with  DBP,  that  to  is  are  gelsolin  and  in  with  the in high  unable  preferentially  concerted  filaments  thought  them  dramatically  The  with  present  actin  to  another  actin  rendering  0  6  into  bind  2  1  polymerization  proteins  actin,  -  where  protein,  monomers,  3  Actin  cells. its  1  blood,  a n d may  8  and  contrast,  clear  the  together  Both  actin.  enters  effects  filamentous  would  1  D  in  plasma  '  Gelsolin,  actin  4  7  with  has  9  have.  2  1  to  g e l s o l i n  find  long,  negative  -  that  of  w i l l  favour  microns  binds  and  proteins  Actin  a b i l i t y  mammalian  actin  importance  1  form  of  the  i t s  interactions  injury  1  that  from  secreted  its  vitamin  p o l y m e r i z e .  binds  reducing action  monomers  of from  circulation. The  by  DBP  the  space.  might  filament).  cytoplasm.  or  quantities  affinity.  the  death  the  the  arises  constituents  called  of  therefore,  major  formation.  circulation  of  with  through  minimize  end  assembly.  of  several  flow  B  g e l s o l i n  importance  of  protein  the  consistency  strength  blood  to  suggested,  associated  disrupt  filaments.  actin  extracellular  ionic  the  of  i n t r a c e l l u l a r  The  to  actin  (binding  has  control  to  of  interactions  calcium  and  of  gelsolin  phosphatidyl  -6-  with  actin  i n o s i t o l .  are The  regulated calcium  concentration a c t i v i t i e s a c t i v i t y  of  in  in  on  the  plasma  is  to  gelsolin  be  from  in  effect is  horse  Of  fluorescence the  of  complementary A n a l y t i c a l l y , absorption; small  biological given  approach  the  are  that  than  component,  commonly  used  can is  the of  several forms  are  control  larger  than  (\iM)  for  assures^  that  protein  in  i s o l a t i o n  of  of  some  which  to  experimental absorption  are  measured study  and  useful offer  i n f o r m a t i o n .  advantages  allows  parameters, be  structure  techniques  redundant has  its  particularly  These  s e n s i t i v i t y  Several  is  severing  study  interest  material  and  the  concentration  This  towards  and  fluorescence  of  calcium  allow  times.  using  greater  would  value  These  physiological  function  a  actin  rather  samples.  sample  as  macromolecules.  its  amounts  This  the  calcium  experimentally  a l l  spectroscopies  study  its The  mM) .  directed  special  being  concentrations  different  polymerization.  plasma  different  requirements. calcium  to  perform  at  the  severing  5  fluctuation  gelsolin  with  of  i n t r a c e l l u l a r  determined  of  interactions  The  (=» 2  role  2  the  cytoplasm.  actin  thesis  approaches.  for  the  affect  w i l l  This  and  of  constant  proposed  plasma  with  This to  each  a c t i n ,  response  gelsolin  concentration  gelsolin the  in  for  stringent  agreement  c e l l .  consistency  the  most  varies  intracellular the  on  v e r t e b r a t e s .  concentration stimuli  the  are  in  varies  g e l s o l i n  with  properties found  required  over  the  study  of  commonly  found  in  characteristic and the  analyzed.  of  a  One  spectroscopic  properties  of  interest. the  a  Careful  protein  different  molecule  analysis  under  The  and  dynamic  spectral  one  several  macromolecules  under  experimental  conditions.  this  the  factors  of  these  B.  Excited  that  probes  is  State  Molecules absorption from  of  process is  to  gives  said  thermal  transfer are  or  deactivation  transition  is  an  to  a  of  by  under  probes  the  with  study  of  variety a  brief  of  review  properties  light.  dissipation. of  states  -8-  processes state.  a  is  include  Radiative photon  the  same  and on or  If  the  product  said  to  be  conversion  states,  depending of  multiplicity.  it  upon  displaced  distinguishable  between  phosphorescence, between  Being  excited  processes  emission  state  several  otherwise  conversion  Overview.  excited  the  chemically  photochemical,  characterized  using  protein  state  An  e q u i l i b r i u m ,  radiative  or  excited  ultraviolet  Photophysical  and  fluorescence  the  by  order.  found  energy,  the  mind,  in  be  of  wide  in  are  rise  to  a  Pathways.  the  knowledge  structural,  allows  Deactivation  visible  photophysical. to  in  thermodynamic  contribute  it  determine  of  fluorescent  b i o l o g i c a l  of  some  protein  deduce  of  characteristics  With  the  obtained  to  properties of  to  with  (perhaps  allows  development  different  attached  combined  study  methodologies)  equilibrium study.  smaller  energy  mechanisms are  termed  whether  the  different  simplified  A states  of  the  illustrates state where with the  the two  same  some  (Figure  version  the  unpaired  same  between  are  of  Jablonski are  paired.  are  (Si,  Transitions  in  etc)  excited  are (T)  between and  where stacks  the  state  allowed  d i f f e r e n t  to  S2,  A t r i p l e t  spin  diagram  arranged  available  states  electrons.  m u l t i p l i c i t y states  pathways  Singlet  electrons  the  m u l t i p l i c i t y  of  3).  of  those is  one  states  of  transitions  m u l t i p l i c i t y  are  spin  forbidden.  Si  . A  So  Figure  Jablonski  3.  The  absorption  mechanical the  order  Diagram.  selection of  10"  16  s.  of  light  rules This  (A)  and is  is  occurs  small  -9-  governed on  a  compared  by  quantum  time  scale  with  the  of time  required  f o r the  order of  10~  displacement  s. As  1 3  of  n u c l e i , which  i s of  a r e s u l t , the molecules are  found i n a  v i b r a t i o n a l l y as w e l l as i n an e l e c t r o n i c a l l y e x c i t e d S  n  (n£l)  (Franck-Condon  solids,  rapid  relaxation  principle). (vr)  to  the  l e v e l o f t h e e x c i t e d s t a t e o c c u r s by  In  the  state  solution  and  lowest v i b r a t i o n a l  g i v i n g up  a quantum o f  e n e r g y a t a t i m e upon c o l l i s i o n s w i t h o t h e r m o l e c u l e s . W i t h few  exceptions,  (n>l), to  will  the  higher  interconvert  lowest  from S i t o  the  So  excited may  radiationless  electronic excited  rapidly  singlet  o c c u r by  mechanisms.  fluorescence  (F)  and  emission of  These  internal  triplet the  (isc) to  s t a t e t o the  forbidden  alternate  deactivation intrinsic the  process the  are  known  conversion  a l s o undergo  manifold.  of  the  as  (ic),  intersystem  Return  i f the  t r a n s i t i o n and  by  from  the  i t is  r e l a x a t i o n mechanism  to  termed includes  emission of a photon.  The  for  (P)  triplet  deactivation  g r o u n d s t a t e i s a s l o w p r o c e s s due  nature  phosphorescence the  the  n  ic)  a photon or  processes  r e s p e c t i v e l y . M o l e c u l e s i n S i may crossing  S  ( i n t e r n a l conversion,  (Si). Further  the  states  of  rate  the  pathways excited  constant  s t a t e are  (kj.) and  d i s s i p a t i o n of the i s defined  (i) that  p a r t i c i p a t i n g processes.  the  In  -10-  an  other  e f f i c i e n c y of  manner, t h e  the  by  each  r e l a t i v e rates  this  to  characterized  compete w i t h  e n e r g y . The  i n terms of  contribute  each  of a l l quantum  yield of  of  fluorescence  molecules  that  which  (Of),  decay  by  represents  fluorescence,  is  the  defined:  (1.1)  Of = kf/(kf+Ikr) where  __kr  groups  deactivation The other an  of  excited In  light,  the  loss  yield,  transient the  mechanisms  studies,  intensity  at  the  lifetime,  spent  molecule  the  the  theory  of  fluorescence transition fluorescence the  time  the  with  the  extent  that  emission  probability  of  the  the  observed  r  i  pulse  of  given  s  within  zero  excited  emission, is  by:  and  is  the  state. the  d i r e c t l y  light  exciting  d -  represents  kf  (Tr),  T  time  the  of  delta  e-t/t  which  emission  a  d(t))  at  in  lifetime  t,  = 1(0)  spontaneous  "allowedness"  However,  compete  after  i n t e n s i t y  fluorescence by  for  molecule.  is  ( 0 )  processes  indicates  Of,  l(t)  I  competing  Si.  quantum  energy  a l l  fraction  as  *  is  the  average  time  T  According  to  probability  of  related  absorption.  defined  2  the  to  The  the  natural  reciprocal  of  transition:  = 1 /  k  lifetime  (1.3)  f  (Tf),  -11-  is  usually  lower  than  the  natural  lifetime  due  T  The  magnitude  pathways  to  determined molecule,  by  surrounding well  as  other  +  f  deactivation  kind  the  other  of  experimental  redistribution  that  may  media,  the  solvent  environment, excited  may  state.  state,  far  and/or  produce  various  from  and  spectral The  be  in  the  terms shifts  or  r e l a t i v e l y hydrogen  long  in  of  and  of  the  is the  medium  pressure,  as  solvent  and  chemical formation  and  condensed to  the  the  as  the  altered, a  relaxed  rates  alterations yields,  state of  are  the often  lifetimes  maxima. with  the  solvent  arise  charge  Frank-Condon  quantum  emission  a  chemical  produce  in  These  is  In  with  formed,  Specific  there  referred  may  changes  the  lasting)  and  state.  strongly  modified the  state  of  different  often  processes.  l i m i t s  bonding  in  i n i t i a l l y  general:  stoichiometric  light,  interaction  of  nature  excited  substantial  interaction  specific  the  interact  deactivating  manifested  result  molecules,  This  alternate  conditions.  absorption, of  of  the  structure  temperature  Upon  properties  of  excited  the  Medium E f f e c t s .  physical  each  state,  its  processes.  (1.4)  the  as  excited  molecule,  of  of  such  competing  Ikp)  efficiency  factors  the  the  = 1 /(k  £  and  the  to  molecule  effects  from  of  approach  s p e c i f i c  interactions charge  may  such  (and as  transfer  complexes. bulk  General  properties  d i e l e c t r i c the  e x c i t e d  such  constant.  mobility  of  The  changes  experiences empirical)  fluorescent case  probe  may  be  usually  B.l.  light, with  an  vector  of  is  the  may  and  affect  lifetime the  on  of  the  observed  a  fluorescent  in  (although  protein  to  the  of  changes to  the  the  (solvent) to  the  structure  In  atoms  in  buffer  by  a  fluorescent  b i n d i n g  experienced  them  medium.  (groups  changing or  of  it  Different  aqueous  fluorophores  by  as  t r a n s f e r r i n g  the  protein  most  studies.  upon  from  probe  o t h e r  fluorophore and  dynamics  interest.  Polarization.  sample  preferential  dipole the  by  the  changes  i s o t r o p i c  there  their  by  index  medium  fluorescence),  extrapolated  Florescence  the  depend  processes.  protein  produced  of  contrast,  affects  detected  the  The  be  protein  If  turn  temperature  macromolecules.  the  a  offered  c o m p o s i t i o n ,  of  to  of  during  useful  be  for  environment  can  in  protein-bound  responsible  molecule  r i g i d i t y  environments  very  may  of  refractive  experienced  be  environments  solvent  bimolecular  different can  in  molecule  which  and  effects,  as  The  the  state,  depolarization  the  solvent  moments  excitation  is  excited  excitation  oriented  of  parallel  (photoselection).  -13-  with the to  polarized fluorophores  the  This  electric  results  in  p a r t i a l l y the  polarized  fraction  of  emission.  light  which  is  Polarization linearly  is  defined  polarized:  p=(I -Ih)/(Iv+Ih)  (1.5)  v  where  I  and  I  p a r a l l e l  and  perpendicular  polarized  excitation.  v  The  the  polarization  molecules lifetime are  are  n  do and  absent.  dipole  not i f  The  polarization emission  w i l l  be  change  other angle  moments  emission  a  between  the  po  PO = ( 3 c o s 0  -i)  observed v e r t i c a l l y  i f  the  absorption maximum  between are  excited  during  depolarization  the  and  6,  maximum  to  p o s i t i o n  extrinsic  p o . The a n g l e  dipoles,  respect  t h e i r  determine  value  i n t e n s i t i e s  with  as  processes  and  or  their  emission i n t r i n s i c  the absorption  related  and  through  the  expression:  2  Commonly  In  6 = 0  Brownian  of  the molecule.  If  to  the  between  polarization rotational  is  (3+cos 8)  (1.6)  2  a n d po = 0 . 5  solution,  interval  /  the  time  of  changes  the  for  In a  steady  is  orientation comparable  and emission, state  molecule  -14-  the  rotation  absorption  observed.  parameters  motion  with  partial  measurements, spherical  the shape  are  related  equation  to  of  the  Perrin.  1/P  <> | is the  the  the  T  hydrated  volume  d i f f u s i o n rewritten  as  a  <J>=  TTV/kT  of  the  a  as  given  by  the  (1.7)  which  i s  related  to:  = l/6Dr  (1.8)  solution,  k  temperature  spherical  shape.  Then  to  i s  and D  is  r  equation  the V  Boltzman  denotes  the  the  rotational  (1.7)  can  be  as:  studies the  of  - 1/3 = ( l / P o - 1 / 3 ) (1+ kTT/TJV)  proteins  correlation  function  of  time  The c o r r e l a t i o n  size  shape  and  molecular  of  the  dimensions to get  i t by  temperature  solution.  possible  time,  according  c o e f f i c i e n t .  1/P  obtain  correlation  absolute of  polarization  6  parameters  the  of  - 1/3 = ( l / P o - 1 / 3 ) (1+  viscosity  constant,  In  2  rotational  molecular  T] i s  degree  time  i s  information  common  measuring and/or provides  molecule.  are  a  practice  viscosity  on segmental  of  information  well  to  polarization  Alternatively,  reasonably  -15-  the  (1.9)  on i f  known,  f l e x i b i l i t y .  i t  the the the is  B.2.  Excited  State  Excited interactions  Quenching.  states with  (n*)  can  suitable  deactivate  "quencher"  upon  molecules  bimolecular (Q)  (Scheme  I)  Scheme  I: kq n*  The  quenching  rate  +  Q  n  constant  (kq)  +  Q  follows  Stern-Volmer  kinetics: On/O  and  according  to  (1.1)  Oo  and  molecule  T, O in  respectively. K  s v  ,  the  the  [Q]  lifetimes  absence is  the  both  according  and  and  (1.11)  quantum  presence  quencher  yields of  of  the  quencher,  concentration,  kq  To  =  constant.  quencher  fluorophore  fluorescence by  the  [Q]  0  the  Stern-Volmer  When t h e of  are  (1.10)  (1.4):  = 1 + kq T  0  [Q]  0  and  T /T  To,  = 1 + kq T  is  at  intensity  " c o l l i s i o n a l  present the  of  a  in  the  instant single  quenching"  to:  -16-  immediate  of  emitter and  vicinity  excitation, may  "static  be  the  affected  quenching"  On/O V  is  as  the  static  the  volume  of  the  after  excitation.  is  been to  the  of  protein.  in  The  the  conformation  alter  the  accessibility  A  the  the  ground  2  7  the  higher  the  within  immediately  measure  of  a  rate  the  Other  fluorescent of  quenching.  excited  state  relative  a c c e s s i b i l i t y  dimensional  structure  of  additives  of  the  or  protein  the  the  quencher.  is  the  of  emitter  quencher  exposed  other under  fluorophore  and  an  ground  state  in  excited  the  is  (excited  dissociative  found  where  may of  a  of  the  proteins  may  study  to  and,  so,  quenchers.  II):  are  complex). and  state.  -17-  such  as  (M)  in  form  a  quencher (N*) i f  complex  they  cases  probe  (Scheme  i f  in  the  excited  excited  dimer),  exciplex is  the  complex  molecule, (excited  quenching  hydrocarbons  state  called  the  the  of  of  luminescent  same  excimer  case  aromatic  transient  to  interpreted  Formation.  special  condensed  a  quenching  presence  the  Excimer  is  three  alter  B.3.  constant  quencher,  be  the  the  more  on  can  by  the  information  fluorophore  that  fluorophore  proteins,  (1.12)  v  surrounding  quenched  equal, the  studies  provide  of  [Q]e tQ]  parameter  sphere  Stern-Volmer  molecule In  a  emission  a c c e s s i b i l i t y things  1 + k q To  quenching  which  The  -  N  is  and  M  are  called  an  different In  this  complexation  it  case, occurs  is the only  In monomer and  a  continuous  and excimer  depend  medium,  the  relative  emission  are  controlled  on t h e concentration  Scheme  of the species  »~ N*  +  M  Pyrene  pyrene  involved.  N +  (monomer emission)  of  diffusion  [NM]*  N +hv  i s  a  Figure  2 8  by  of  II:  N  excimers.  intensities  in  (excimer [exciplex] emission)  classical 4  shows  M+ hv  example  of molecules  the fluorescence  that  form  characteristics  solution.  400  500 w a v e l e n g t h (nm)  Figure 4. Emission Properties of Cyclohexane. (a) Emission spectra B)7.75xl0C) 5 . 5 x l 0 D) 3 - . 2 5 x l O (b) F l u o r e s c e n c e response of pyre cyclohexane. Excitation pulse, p Excimer, fc(t). 3  3  2  Pyrene Solutions i n of pyrene: A) 1 0 " M ' M E) 10 M G) I O M. ne (5 x IO" M) i n ( t ) . Monomer, f ( t ) .  _ 3  8  -18-  2  _  3  -  3  M  4  In  steady  (^10 *M)  increasing  a  to  time  (figure  maximum  of  the  4a  In  continuous  a  the a  increases  monomer).  shows up  that  monomer the  of  concentration,  wavelengths  of  measurements  fluorescence  4  the  state  the  is  concentrations  predominates.  band  intensity  appears  normalized to  while  the  the  excimer  decreases  that  the  4b) .  of  of  intensity monomer  shows  (Figure  characteristic  longer  to  domain,  With  at  relative  time-dependent  then  excimer  broad  been  decrease  and  low  monomer  in  has  at  a  build  The  rise  excited  state  reactions. In  studies  derivatives, of  of  proteins  excimer  labelling  and  covalently  formation  chain  is  dynamics  labelled  controlled rather  than  with  by  pyrene  the  by  sites  diffusion  control. Studies synthetic in  some  of  polymers cases  fluorescence  also  in  surfaces  3  9  -  3  in  the  3  in  state,  the  state.  in  the  or  case  repulsion ground  the of  the  state.  Such  '  3  have  5  media  and  Ground  pyrene  that  chains  exhibited  of  state  adsorbed In  3 9  between  dimer-excimer  to  brought the  helps  to  some  shown  can  that  interact  excimer-like the  complex  complexes  such  linking  paracyclophanes,  generally  4  excitation  paracyclophanes.  hydrocarbon  3  aqueous  seems t h a t t h e r i g i d i t y o f t h e p y r e n e s surface  attached  p r o t e i n s ,  ground  with  in  covalently  and  from  ground  and  8  3  pyrenes  detected 6  -  arises  the  been  2  the  hydrophobically  formed  pyrene  to  some  have s o l i d  systems, about  by  it the  chromophores, overcome  molecules  transitions  in are  the the not  uncommon  phenomena,  but  differ  mechanism  of  excimer  or  dimer-excimer  certain  of  their  B.4.  monomer-excimer  Excitation  Under (D*)  may  molecule state  transfer  D*  (acceptor)  Scheme  Energy  more  described  processes  absorption  and  may  above.  be  emission  circumstances,  i t s  energy  resulting  (donor)  and  (Scheme  III).  This radiative  energy or  in  to  by  characteristics.  the  the  excited  state  ground  state  another  quenching  e x c i t a t i o n  +  A  reabsorption mechanisms  transfer  nonradiative  radiative  have  Monomer-  distinguished  of  of  the  the  D  +  excited  quencher  A  A*  A  donor  studied  III D*  or  widely  Transfer.  appropriate  (A),  from the  mechanism of  the  involve  excitation  been  proposed  processes; i f  the  light  the  and  between  of to  it  and is  emitted  by  -20-  the  may  D.  of  A  a  be  by  t r i v i a l  occurs  by  Radiationless  de-excitation  acceptor.  explain  A  called  excitation  simultaneous the  D  + hV  Several  of  the  mechanisms  nonradiative  energy  transfer.  These  transfer)  and  Forster the  4  include electron  derived  0  rate  of  acceptor  energy  between  donor  between  transfer  and  (E  is  is  the  50  distance  %.  The  overlap  possible  to  suitable  donor  map  divided  of out  in  two  major  covalently  or  are  of  l )  1  '  The  donor  and  relationship  and  the  distance  described  by:  (1.13)  6  transfer  highly  efficiency  dependent  acceptor in  relates  pair.  proteins  on  Thus,  by  it  selecting  the is a  pair.  to  Study  probes  I n t r i n s i c  fluorophores  donor  is  energy  is  distances  Probes  components  the  Ro  the  luminescent  e x t r i n s i c : natural  of  acceptor  Luminescence  The  which  value  spectral  C.  at  (E)  .  dipole-dipole  i s o l a t e d  (R)  -  - 1  that  from  parameters.  sites  R = Ro Ro  an  efficiency  acceptor  resonance)  expression  originating  observable  (resonance  (inductive  quantitative  transfer  and  interactions  exchange  interaction pair  between  a  energy  coulombic  coulombic  Proteins.  used  categories  to  study  (Figure  fluorophores the  purposely  noncovalently  native added, bound,  -21-  5) :  are  protein such  as  proteins  can  Intrinsic  those  that  be and are  while  extrinsic  organic  molecules  and metal  ions.  Luminescence  probes  to  study  covalent noncovalent ions (lanthanides)  tyrosine  tryptophan  F i g u r e 5. Proteins. C.l.  Schematic  Intrinsic  The  are  the  This  of  A  14  comparable e f f i c i e n t  effect  the  size  is  proteins  lacking with  s e n s i t i v i t y quantum  is  mainly  of  the  Study  due  to  native  The  as  fact  the  p r o t e i n .  in  amino  emission  is  also  from  the  acid some low  phenylalanine,  due  to  i t s  c o e f f i c i e n t  tryptophan  is  Tyrosine  in  observed  -22-  which  results  nearby  and  distance  and  absorption that  the  protein  Emission  not  and  other  unfolded  Forster  in and  tryptophan  transfer,  the  tyrosine. usually  the  proteins  by  tyrosine  dominates  both  many  tryptophan,  (defined  yield).  contain  quenched  in  free  contrast,  u s u a l l y  that  in  also  since,  in  to  chromophores  tryptophan,  tyrosine-tryptophan  residues  compared  is  transfer  fluorescence  acids  proteins  for to  Probes  fluorescent  Tryptophan  of  tyrosine. «  Luminescent  occurring amino  p h e n y l a l a n i n e . fluorescence  of  Probes.  naturally  proteins  metal  Diagram  proteins  emission  low times  is  very  sensitive ideal  environmental  probe  However, of  to  to  the  fact  .tyrosine  and  C.2.  of  many  the  probes  sensitive  have  been  detection  of  Probes.  The  extrinsic  ideal  of  properties.  identifiable  that  the  structure  is  (except  produce  a  simple,  stable  and  always  possible  experiment. structure binding before  the  C.2.a. advantage bound  the  should  the  relevance  of  a  of  using  fluorescent  the  the  In  provide  selective  environments.  occur  should in  a  its  three  of  protein  results  Probes  to  is  that  can  be  Study  -23-  a  almost  dimensional a  be  result ruled  of out  assessed.  Proteins.  bound  mild  is  in  particular  as  must  so  Excitation  a  three or  a  dimensional  it  for  procedure  easily  monitored  practice,  the  have  selective  achieved  suitable  noncovalently  probes  extrinsic  probe  should  probe  to  Noncovalent  make  be  labelling  probe  can  which way.  changes. number  handle).  D e s t a b i l i z a t i o n the  in  an  Several  their  be  it  large  spectroscopic  sensitive find  in  to  should  intact  signal  to  during of  and  left  for  should  developed  changes  a  residues  fluorescence  manner  protein  contain  d i f f i c u l t .  Binding  make  conformational  proteins  results  should  4 1  tryptophan  Extrinsic  number and  that  protein  and/or  interpretation fluorescent  study  changes  over  labelling  The  main  covalently procedures  can  be  used.  the  probe  A  is  property  disadvantage  not always  measured  One  example  is  possible  may r e f l e c t of  that  the  t o be  bound  a noncovalent  exact  determined.  and free  probe  to  a  exhibit  large  fluorescent  class  solvent  i n water  blue-shifted proteins large,  of  or  in  e-g.,  water  compared  extensively  their  wide  use  in  that  effects.  practically  non-  investigation  molecules.  A  state both  state  has  (So—>S_) states  promote  being  the  transitions the have  been  naphthalene  of 4 2  found  the  solvents  also  in  the  of  So.  only  by  Specific  transitions  derivatives.  -24-  to  these in  the  singlet  (Si—»Si  high  c t  ) ,  polarity and  However,  the  and  led  of  the  t r a n s i t i o n  to  environment.  have  relaxation  state  in  changes  systems  transfer  are  = 0.006  These  of  to  changes  Of  Excitation  but  not  is  and  adsorbed  photophysics  t r a n s f e r  controlled  of  MANS  solvent  charge  enhanced  when  ethanol.  the  d e a c t i v a t i o n  are  viscosity  a  emissive  charge  r a d i a t i o n l e s s  of  is  observed  of biological  picture  to  and  The of  in  emerged.  leads  are  fluorescence  solvents  =0.50  intensive  excited  They  yield  studies  complex  is  derivatives  but their  Of  used  anilinonaphthalene  quantum  to  the  MANS  macromolecules.  the  Also  (MANS).  nonpolar  other  of  fluorophore.  2-(N-methylanilino)naphthalene-6-sulfonate belongs  location  i t s these  polarity  but  solvent  effects  of  the  by  aryl-  Scheme  IV:  C.2.b. Several label  Molecules  fluorescent  organic  particular  amino  advantages  over  position  labelling  the  of  primary  that  Bind  reagents  acids  noncovalent  structure  Covalently  i n  a  that  reacts  few reports  in  proteins  in  the  majority  reactivity  of  in  of  means  the three  Metal  europium,  have 4  4  '  4  5  with  study  Ions. been  These  to  few,  the  the location  exact i f  probes  fluorescent  although or  that  PIA  a  there  cysteines  the high  excimers, of these  i n t r i n s i c degree  attached offering amino  of the protein.  mainly  extensively  i n  have  low  a  very  are  histidines  the  plus  The l a n t h a n i d e s , used  a  covalently  form  structure  i s  methionine  gives  derivatives  are able  (PIA)  The f a c t  are  thiols,  dimensional  C.2.C.  p r o t e i n s .  to  that  offer  can be determined  cysteines,  proteins  Pyrene  sometimes  excellent  with  cysteine.  cysteine  s e l e c t i v i t y . proteins  mainly  lack  They  to  known.  of PIA reaction  that  i n  Proteins.  developed  proteins.  probes  N-(1-pyrenyl)-iodoacetamide probe  have been  i n the protein i s  to  4  of to an  acids  3  terbium and studies  of  sensitivity  (low  absorption  coefficient  observe  their  luminescence,  using  laser  or  a  transfer. they  are  calcium of  these  In  with  proteins,  sufficiently by  a  terbium  probes  to  in  but  high  they  can  be  conventional  tryptophans close. some  study  quantum  The  has  binding  -26-  To  directly  through  suitable  isomorphous  proteins  calcium  excited  lamp  are  yield).  energy  donors  i f  replacement  of  encouraged sites  in  the  use  proteins.  R E S U L T S AND  PART  I.  Isolation  In  isolating a  ideal  method of  in  simple  a  three  is  and  protein  as  properties,  vitro  of  the  the  during  manner  to  of  the  etc.  preserving In  c r i t e r i a .  stages  by  the  practice,  properties  The  The  of  the ionic  along  some  molecular  an  quantities  interactions,  followed as  large  these  molecular  such  selected,  protein.  among  heat,  are  is  while  physicochemical  protein,  was  f i r s t on  4  6  -  5  plasma. ' 3  1  development  '  4 6  isolation been  preparative platelets ternary  '  activity of  5 2 - 5 4  and  reported  is  Several  which  methodologies. of  various  complex  that  rabbit  that  various  also  other  in  the  recognized  weight  or  macrophages  polymerization.  i d e n t i f i e d  Gelsolin  1 3 - 1 6  detected  actin  required  subsequently  c e l l s .  have  source  should produce  s o l u b i l i t y ,  effects  provided  its  on  a  Plasma.  i n  activity.  its  was  after  balance  process  Gelsolin  it  a  s t a b i l i t y  p u r i f i c a t i o n property  protein,  structure  depends  such  from Horse  inexpensive  always  separation  Gelsolin  preparation  dimensional  there  to  of  DISCUSSION  in  found  c o u l d be  a  variety  as  physical be  occurs -27-  used  a  used  by when  to  has  taking it  protein  been  used  of  for  further  isolated  to  in  gelsolin  advantage  binds  It  vertebrate  optimize  been  follow  schemes.  secreted have  effects to  of  properties  Gelsolin  species  These  purification  procedures  can  1  due  from  of  the  actin  and  DNAase  1.46-48  column been  gy  containing  attached,  single  a  step.  transient,  In  but  some  However,  this  is  the  reported  binding  is  to  a  a v a i l a b i l i t y isolation the  based  gelsolin  method  method  plasma  resulted  details)  and  in  calcium  of  an  The  or  by  of  the  Bryan  steps.  5 6  was  6)  for  -20  gelsolin  charge  of  takes the  ions.  -28-  a  as  The  5  the  factor  The of  upon  to  include  by  by  various  of  of of  human  supernatant  blood  advantage  protein  low  modification  horse  used.  its  i s o l a t i o n  experimental  u n t i l  5  isolation  prepared  (see °C  is  the  of  '  by  plasma  exchange  centrifugation  at  3  of  the  the  Another  gelsolin  followed  (Figure  actin-  procedures  for  only  that  l i m i t i n g  Other  here  plasma  of  a  interactions  sulfate,  ion  plasma  specific  in  method.  separate.  components  anticoagulant  stored  surface  the  this  a had  is  form the  a n t i b o d y .  is  strategy.  presented  from  purification the  of  I  obtained  by  to  i s o l a t i o n  ammonium  horse  gelsolin.  of  use  DNAase  actin  to  i t  through  disadvantage  d i f f i c u l t  the  this  reported  presence  the  on  with  from  the  is of  purify  antibodies  chromatographic  The  that  such  f r a c t i o n a t i o n  column  the  on  which  added  g e l s o l i n - s p e c i f i c  of  precipitation  to  advantageous based  be  suffers is  to  presence  could  order  complex  extract  p u r i f i c a t i o n  the  actin  in  platelet  support  yield  technique  actin-gelsolin  a  inert  plasma,  complex  where  an high  gelsolin  method  passing  in  the  section  for  method  for  the  change  binding  to  1 1 thawed plasma in the presence of 3 5 mg P M S F  dialysis  against  (3 c h a n g e s i n 3 days)  2 5 mM T r i s - H C l -- I 0 . 5 mM C a C l IpH 7 . 5  c e n t r i f u g e 10000 x (10  add NaCl  mix  to  (settled  (discard)  21  volume)  DEAE-Sephadex equilibrated  A-50  against  2 5 mM T r i s - H C l l C_-^. 0 . 5 mM C a C l 5 0 mM N a C l pH 7.5 2  stir (2 h )  (continue  g  minutes)  3 5 mM  with  2  next  page) -29-  filtrate  retentate (regenerate DEAE-Sephadex)  a d d EDTA and  ' 2 5 mM T r i s - H C l 1 mM E D T A • 5 0 mM N a C I ImM N a N .pH 7.8  •  to  adjust  1 0 mM pH t o  7.8  apply to column ( 3 1 x 6 cm) packed with DEAE-Sephadex A-50 e q u i l i b r a t e d against:  3  e l u t e w i t h a 0.05 t o 0.3 NaCI l i n e a r gradient  M  collect fractions containing g e l s o l i n and concentrate with a m m o n i u m s u l f a t e a t 60 % saturation  pure (yield  Figure  6.  Isolation  of  Gelsolin  -30-  gelsolin 30-40  from Horse  mg)  Plasma.  The the  treatment  of  the  presence  of  calcium  purification  of  gelsolin  in  the  are as  filtrate  retained a  batch  by  in  faster  h vs  obtained the  the  by  absence  a  ion  17 h)  amount  ion  exchanger  contaminant liters  or  by  the  followed  is  proteins  more,  Proteolysis and  that  a  was  used,  in  The  5-15%  gradient  electrophoresis,  using  the  reduce  5 7  the  and,  by  viscosity  of  F-actin  -31-  simpler  and  eluting  the  is  exchanger with  a  on  the  step.  The  level  of  in  NaCI  more  used  yield  obtained.  a l l  is  steps  is  at  scheme  polyacrylamide  gel  the  out  when  purification  buffer  monitoring  plasma  quantity  performing  PMSF.  as  anion  calcium  the  the  carried  depending  greater If  a  Laemmli,  and  emerges  purification  the  reproducible  minimized by of  to  the  the  of  in  of  Gelsolin  step,  Further  exchanger  degree  same y i e l d  varies  retained.  highly  presence  using  used  step.  This  cations  profile  high  considerably  protein  divalent  elution  exchanger  was  anion  components  the  perform.  the  The  ion  the  an a  single  of  but  gradient. of  a  exchanger.  to  adsorbing the  in  with  permits  provided  column,  of  ions  most  procedure,  conducted (3  while  plasma  system ability samples.  developed of  2  4  °C  was  by  gelsolin  to  PART  A.  II.  Physicochemical  Absorption  The  model  Coefficient.  difference  between  solution BP-2000V  gelsolin  Characterization.  i n  refractive  and solvent d i f f e r e n t i a l  solutions  o f known  index  at  was measured  5 4 6 nm, with  refTactometer  absorbance  (A)  ,  a  Vir-Tis  a  set  f o r  at  (An)  2 8 0 nm  of  (Table  I) •  An  A  sample 1 2 3 4 5 6 7 8  0 0.304 0.587 0.608' 1.175 1.215 1 . 7 62 2.430  0 0.291 0.570 0.593 1.309 1.286 2.117 3.296  Table I. Calculation of the Absorption Coefficient of Gelsolin. Difference i n refractive index (An) between different g e l s o l i n s o l u t i o n s o f known a b s o r b a n c e (A) a n d solvent. Buffer u s e d : 2 0 mM T r i s - H C l - 1 5 0 mM K C 1 - 1 mM EGTA - (pH 7.5) . See t e x t f o r d e t a i l s .  A  value  increment Handbook at  pH  proteins plot  of  0.186 ml/g  (dn/dc)  f o r  gelsolin  of Biochemistry 7.0  and  l i s t e d ) .  of A against  that 5  8  was assumed (a  f o r bovine  shows  l i t t l e  Measurement  serum  a  refractive  quoted albumin  variation  of the slope,  An a n d a p p l i c a t i o n  -32-  value  as  i n i n  among  dA/dAn,  of the result  the water the of  a  A =elc,  allows for  calculation  horse  plasma  Polyacrylamide  The 90  kDa  absence  of  composed linked  by  (vide PAGE  value  for  infra!. known None  the  (Figure  a b i l i t y  by  .  Dodecyl  as  a  in  the  suggesting  the  as  value  to  S u l f a t e  single  band  of  presence  that  and  gelsolin  opposed  and is  method)  to  and  behavior  glycoproteins  is  subunits  primary  binding.  affecting  This  the  -33-  been  which  to  mass  on  r a t i o .  "  SDS  modified For  attributed  effect  4  the  gelsolin.  may  1  velocity  p a r t i c u l a r  structural  charge  with  proteins  to  the 3  chemically  has  in  sources. '  compared  some  and  structure  the  reported  corresponds  of  molecular  sedimentation of  migration  properties  kDa  cytoplasmic  anomalous  f i l t r a t i o n  cases  90-93  the  same  plasma  anomalous  SDS of  by  these  its  7),  polypeptide  abnormal  of  chemical  normal  gel  for  proteins,  determined with  this  The  nm  280  (SDS-PAGE).  migrates  comparable  other  by  - 1  at  bridges.  (obtained  obtained  proteins.  the  is  However,  is  other  single  disulfide  literature  value  gelsolin  cm  - 1  Sodium  by  5 7  coefficient  ml mg  1.4  Electrophoresis  SDS-PAGE  a  values  16,47-54  £=  mercaptoethanol,  of  This mass  Gel  %  8  absorption  Weight  purified  on  the  gelsolin,  M o l e c u l a r  B.  of  to  protein interfere may 6  0  have  Figure  7.  plasma  Molecular  g e l s o l i n ,  determined series  to  of  least plasma  C.  in  Amino  The horse 739  Acid  amino  g e l s o l i n .  6  is  phosphorylase  in  lane  a b  are was  gel in  Da  was  using  a  indicated  serum  line  Horse  symbol,  90000  bovine  s o l i d  Inset  of  masses  BSA,  The  SDS-PAGE.  square  mass  1,  acid  in  albumin.  LDH,  determined  that  lane  2  shows and  by  horse  purified  3.  composition  gelsolin  acid 1  a  by  Composition.  amino  plasma  80908.  analysis.  gelsolin  by  Molecular  Abbreviations:  lane  Gelsolin  molecular  dehydrogenase. square  plasma  of  indicated  have  standards.  parentheses. lactate  Mass  Such  was  residues, a  protein  Calculations  based  presented  scaled  to  the  same  would upon -34-  have this  in  achieve as  for  molecular  table a  II  for  protein pig  plasma  weight  composition  of  Mr  suggest  = a  p a r t i a l  s p e c i f i c  hydration  level  amino  6  3  acid  volume (8)  of  (V)  6 2  0.393  of  g water/  horse  0.727 g  cm /g,  and  3  protein.  p i g  6  1  human  asx  66.6  73  74  thr  43.5  38  41  ser  61.5  46  44  glx  84.6  89  92  pro  38.6  39  38  gly  65.7  67  71  ala  62.4  66  74  val  64.4  60  58  met  7.6  12  12  ile  22.1  24  23  leu  55.8  55  57  tyr  21.8  23  22  phe  30.7  31  31  lys  43.9  44  45  his  14.5  13  13  arg  35.3  39  40  cys  n.d.  5  5  trp  n.d.  15  15  TOTAL  739  739  755  a  Table II. Amino A c i d Composition of Plasma (n.d.) not determined. (a) a s s u m i n g 5 c y s a n d 15 t r p r e s i d u e s p e r molecule.  -35-  a  6  Gelsolins. gelsolin  D.  Sedimentation  In  a  density to  Coefficient  solution  than  macromolecules  the solvent,  centrifugal  behind  i n the upper  pure  solvent.  This  Schlieren  optics,  refractive  index  photographic  containing  force. part  As they of  at  the vessel  i s  gradient.  them  a  can  based  on  The image  defined  i f  sediment  gradient  which  plate  having  the macromolecules  the bottom of the c e l l  strong  of  of  time  a  w i l l  they  region  can be  intervals  imaging recorded (Figure  a  leave  using of  a  on  a  8).  Plateau Region  m  , Solukionifj(AtT Meni-I  dn dr  to  consisting  detected  the  Solvent Boundary Region Region  sediment  subjected  down,  be  higher  Solvent — Air Meniscus  •n  Schlieren Peak] t _ Base Line  | "t-Top of Cell  Bottom of Cell -J  inner Reference Ed_e  j  Outer Reference Edge  Direction of Sedimentation  Figure  8.  The is  Typical  radial  measured  inset)  yields  Schlieren  distance,  and a a  plot  slope  r, of  equal  Pattern.  65  traveled In  r  against  to w s: 2  -36-  by the Schlieren time  (Figure  peak 9,  s  =  (1/w )(dlnr/dt) 2  where: s  =  sedimentation  w  =  angular  r  =  distance  o f sedimenting  rotation  (cm)  t  =  time  coefficient  velocity  (sec)  (rad/sec) boundary  from  center  of  (sec)  S20,w = (S) (Tl_,soln/T|20,w) ( [ l"Vp] 20, w/ [ l"Vp] , soln) T  5  T  0 4 0 Figure The  9.  . Absorbance at 280 nm Intrinsic  i n t r i n s i c  determined  t o  sedimentation Inset  , 1  Sedimentation  Coefficient  sedimentation  coefficient  be  extrapolation  4.8 S  coefficients  i s the data  by  (S20,w) t o  f o r determination  -37-  o f  zero  o f  Gelsolin.  gelsolin of  was  determined  concentration.  of one value  o f S20,w  The depend  on  calculate  sedimentation  coefficient  concentration. the  For  intrinsic  this  is  reason  sedimentation  S°20,w  "  generally it  is  found  to  desirable  to  coefficient,  S  20.w-  S o,w  Um  2  c-^o Figure against  9  is  sample  obtained  E.  presence  Molecular  plot  of  absorbances.  by  S20,w s°20,w  e x t r a p o l a t i n g  concentration. and  a  This  of  value  was  values  for  4.8  10~  =  the  x  g e l s o l i n  r e s u l t s  obtained  both  in  sec  1 3  was  to the  zero absence  calcium.  Weight  and  Stokes  Radius  1  Determined  by  Gel  Filtration.  Determination by  gel  f i l t r a t i o n  technique to and  is  separate Stokes  carried substance globular  gel  on  radius  1  by  of  the  according  of  with  gel  by  values  weight 6 6  f i l t r a t i o n Molecular  gel  elution  the  molecular documented.  size.  determination  interest  10  well  to  some  and  is  a b i l i t y  comparing  protein  radius  chromatography  based  accessible  details),  Stokes'  molecules  out  Figure  of  media weight  f i l t r a t i o n  parameters  obtained  The  of  for  is the  several  standards.  shows to  versus  the  the log  plot  of  protein, of  Kav see  molecular -38-  (the  average  experimental  weight,  Mr,  for  value part a  set  of for of  standards t o o b t a i n by i n t e r p o l a t i o n of  the molecular  weight  g e l s o l i n equal t o 75000. As d e s c r i b e d by Laurent  Killander,  6 6  a p l o t of (-log K a v ) / 1  2  and  versus Stokes' r a d i u s  (Rs) o f the same standards used t o c a l c u l a t e the molecular weight  p r o v i d e s a c a l i b r a t i o n curve f o r the determination  of Rs o f g e l s o l i n . Rs = 3.8 nm plasma  gelsolin  i n the presence  was c a l c u l a t e d and absence  f o r horse of calcium  (Figure 11).  F i g u r e 10. Determination o f the M o l e c u l a r Mass of Horse Plasma G e l s o l i n by Gel F i l t r a t i o n . Standard p r o t e i n s were d i s s o l v e d i n 20 mM MOPS - 0.5 mM C a C l - 150 mM KC1 (pH 7.0) and e l u t e d with the same solvent from a column (1 x 30 cm) o f Superose 6 HR 10/30. The square i n d i c a t e s the p o s i t i o n o f horse plasma g e l s o l i n . The s o l i d l i n e was determined by l i n e a r l e a s t square a n a l y s i s . 2  -39-  The  value  f i l t r a t i o n  from  calculate  the  the  the  can be  (s°20,w)  to  of  combined  radius  with  sedimentation molecular  following Mr  Stokes'  (Rs)  determined  sedimentation v e l o c i t y  weight  (Mr)  of  by  gel  coefficient  experiments, gelsolin  to  according  relationship:  = 6JCT1 R s N s ° 2 0 , w  /  d  _  V p) = 7 5 0 0 0  where: N  = Avogadro's  T| = v i s c o s i t y  number of the  p  = density  of  V  = partial  specific  0.9  the  by  used  standard  least  i n  The  gelsolin. square  of  Gel F i l t r a t i o n . as  proteins  Pharmacia. plasma  volume  of  the  protein  Stokes Radius (na)  11. Determination  Gelsolin were  solution  T  0 Figure  solution  figure was  The 10.  assumed  square The  Stokes'  to  indicates  s o l i d  line  analysis. -40-  Radius  same The be  10 of  Horse  conditions Stokes' those  the was  and  r a d i i  determined  buffer of  indicated  p o s i t i o n  Plasma  of by  the  by  the  horse linear  Direct of  gelsolin  use  of  that, The an  identity  mass  for  (from  1.3.  In  to  consistent  it  with  by  experiments masses  SDS-PAGE  or  a  6 1  be  the  SDS-PAGE  Implicit  in  is  the  Mr the  assumption proteins.  obtained  suggests  that  case  of  from  hydration, or  amino  the  and  acid  f r i c t i o n a l the  gelsolin.  anhydrous  sequence  ratio,  rather  =  1.2  f r i c t i o n a l  This  than  the  data)  f/fo  coefficient  sphere.  protein  of  molecular  experimental  f r i c t i o n a l  Further  use  the  such  of  the  value  an  is  elongated  noted  that  somewhat  the and  lower,  for  value  of  Mr  =  sedimentation  but  comparable  gel sol i n s  of  velocity  to  known  75000  molecular  amino  acid  6 4  for  does  of  the  denatured  not  either  molecular gelsolin by  velocity  somewhat  used.  weight  with  gel  value  that  trimers  The  value  overestimated. -41-  or of  obtained  obtained  f i l t r a t i o n ,  experiments,  form dimers,  conditions is  the  globular  comes  is  f i l t r a t i o n  protein,  gelsolin  an  globular  gel is  '  the  10,  estimate  for  the  its f  75000.  only  of  to  one.  sedimentation  under  is  were  Comparison  native  degree  calculated  sequence.  in  of  conclusion  fo  should  obtained  values  relation,  filamentous It  two  data  figure  hydrodynamic  and  i f  valid  calculate  this  molecule  in  be  a  the  either  gelsolin  value  warranted  such  radius,  1  coefficient  or  the  is  a  curve  w i l l  of  f i l t r a t i o n  gives  estimation  Stokes  gel  standard  assumption  for  of  also  the  evidence  -  use  by  with  the  chromatography,  demonstrates higher 90  This  kDa has  that  aggregates obtained been  by  found  a l s o f o r t h e o t h e r g e l s o l i n s o f known amino a c i d T h i s o v e r e s t i m a t i o n may the  protein given  occur  be  by  due  t o an  i t s primary  sequence.  i n t r i n s i c property structure.  This  of  would  i f some s t r u c t u r a l f e a t u r e i n t e r f e r e s a t t h e b i n d i n g  s i t e s o f SDS,  a f f e c t i n g t h e m i g r a t i o n o f t h e complex i n t h e  gel.  F.  Ultraviolet  Absorption  and  Fluorescence  Emission  Spectra.  The similar  absorption to that  of  spectrum  of  most p r o t e i n s . The  c o n t a i n s a s i n g l e maximum a t 280 tryptophan  gelsolin  residues  absorb.  A  nm  region  at  a t t r i b u t e d t o the absorption of tryptophans uncorrected  fluorescence  gelsolin  i s maximal  at  328  nm  tryptophans  obtained  at  absorb),  295  tryptophans.  This  nm  by  result  can  (Figure  be  nm  be  intensity  of  inset) .  The  alone.  12  (where t y r o s i n e s  the  is  known  environment.  4 1  from  to  320  nm  The 350  to  be  very  on  efficient which are  i n close p r o x i m i t y i n the n a t i v e p r o t e i n .  emission  depending  on  sensitive  the  that  of  the  energy expected  Tryptophan  w a v e l e n g t h o f maximum e m i s s i o n nm  nm  can  excitation  t r a n s f e r from t y r o s i n e s t o tryptophans, t o be  nm  superimposed  selective shows  is  240-300  290  emission  s p e c t r u m o b t a i n e d on e x c i t a t i o n a t 280 and  12)  where t h e t y r o s i n e and  shoulder  The  (Figure  polarity  to  the  varies of  the  solvent.  I t has been r e p o r t e d t h a t g e l s o l i n  tryptophans. ' 6 1  the  6 4  tryptophans  possesses  The e m i s s i o n o b t a i n e d i s an a v e r a g e o f a l l i n their  different  environments  protein.  However, t h e v a l u e o f m a x i m a l e m i s s i o n  suggests  that the bulk o f the tryptophans  hydrophobic  15  i n the  a t 328 nm  are i n a  fairly  environment.  F i g u r e 12. A b s o r p t i o n S p e c t r u m o f G e l s o l i n . G e l s o l i n (1.9 mg/ml) i n a 2 mm c e l l i n 20 mM MOPS - 150 mM KC1 - 1.0 mM EGTA pH 7.6. I n s e t : f l u o r e s c e n c e e m i s s i o n s p e c t r u m o f g e l s o l i n (0.035 mg/ml) i n t h e same b u f f e r w i t h e x c i t a t i o n a t 280 nm. a.u. «= a r b i t r a r y u n i t s .  -43-  G.  C i r c u l a r Dichroism  Circular studying  Spectra.  dichroism  protein  (CD)  conformation  r a n g e o f c o n d i t i o n s . The i s used to study the chain  208-240 nm  as  of  legion.  the  structures to  3 sheet  technique  s o l u t i o n under region of the  chromophores  effects in this  s p e c t r a f o r model p e p t i d e s , each  in  useful  region.  Figure  i n d i c a t i n g the the  spectrum  than 13  wide  (main amides  shows  CD  c o n t r i b u t i o n of  ellipticity  s t r u c t u r e s show a  h e l i c a l s t r u c t u r e s show two  other  for  a  secondary s t r u c t u r e of p r o t e i n s  conformations),  have m i n i m a l  is a  i n the  s i n g l e minimum  minima a r o u n d 208  and  222  far  UV  and  a-  nm.  80  190  Figure  13.  210  Polypeptide  (2) (5-structure and  230 ,  X  250 (nm)  Reference Data.  (3) random c o i l  100%  (1) a - h e l i x ,  i n t h e 200-240 r e g i o n .  6 7  The (figure helix, The at  f a r 14)  U V  c o i l  residue  at  2  of  significant  horse  -7800  value  plasma  gelsolin  contributions  and ^-structures  e l l i p t i c i t y  2 0 8 n m , [6]208, i s  cm /dmol  spectrum  suggests  random  mean  CD  in  the  native  calculated  d e g cm /dmol 2  from  a-  protein.  for  gelsolin  a n d [9]222 = - 6 1 0 0 d e g  222 nm.  T  X (nm) Figure  14. Far  Gelsolin  pH  7.5.  as  t h e mean  c e l l .  UV C i r c u l a r  ( 0 . 6 mg m l  The  - 1  spectrum  T h e mean  )  residue  residue  in  was  Dichroism  2 0 mM M O P S , measured  e l l i p t i c i t y  weight  of horse  -45-  in  Spectrum  of  Gelsolin.  1 5 0 mM K C 1 , 1 mM E G T A a  0.5  mm  was c a l c u l a t e d plasma  pathlength using  gelsolin.  110  Upon and  [0]222  those but  addition  -5600  =  presented are i n  cm /dmol, obtained [6]222  values  t o  33000  o f  The  The  gelsolin  2  content  gelsolin.  tryptophan  7  from  radiationless tryptophan and  excited  decrease maximum gelsolin  deactivation altering  states.  That  i n intensity emission.  calcium, value  an  error  e l l i p t i c i t y  19 % .  at  t h e energy  of  unfolding nm t o  2 95  studies  of  avoid  o f  free  increases  the  excited  state  gapbetween a  40  o f  ground  continuous  t h e wavelength  o f maximum  approximately  as the  fluorescence  produces  altering  7 0  as well  temperature t h e  o f  Denaturants.  Thermal  i s , heating  until  8  deg  i n t h e absence  t h e thermal  The wavelength  i s constant  be  shows  emission  o f  without  o f  6  nm.  was performed  that  -40430  =  t o  model  a n dC h e m i c a l  follow  shown  without  l i k e l y  o f gelsolin  the tyrosines.  have  1  2  o f the tryptophan  t o  Excitation  interference  2 2  g e l s o l i n ,  The e l l i p t i c i t y  9  222  o f maximum  used  [8]  t o be approximately  intensity  were  i s  deg cm /dmol a t  wavelength  6  peptide  t o Temperature  fluorescence  are similar t o  a n d absence  a l .  2  a-helical  Stability  et  d e g cm /dmol  c a l c i u m was c a l c u l a t e d  H.  t h e values  % a-helical  100  values  b y D o i e t a l . f o r p i gplasma  i n t h e presence  -40430 a  2  These  2  by Kwiatkowski  =  because  d e gcm /dmol.  contrast  both  2  [0]208 = -6400 d e g c m / d m o l  o f calcium,  emission  °C ( f i g u r e  o f i n 15).  At  higher  starts 350  temperatures,  to  shift  nm.  The  unfolding the  of  aqueous  towards  the  observed gelsolin  and  intensity  with  expected  increased  rate  for  tryptophan. with  suggesting  that  45  red  the  until  The  there  midpoint is  l i k e l y of  the  half  in  the  a  a  value  due  to  tryptophans in  (Figure  is  wavelength  reaches  radiationless  However,  gelsolin  it  decrease  temperature of  emission  are  exposure  environment.  coincides  maximum  changes  emission  free  the  15)  decay break  at  the to  tryptophan shows  the  discussed point  wavelength  unfolded  of  that  s h i f t ,  approximately  °C.  F i g u r e 1 5 . Thermal D e n a t u r a t i o n o f G e l s o l i n as F o l l o w e d by Fluorescence. Gelsolin unfolding ( 0 . 0 3 5 mg/ml) i n 2 0 mM M O P S - 1 5 0 mM K C 1 - I m M E G T A (pH 7 . 2 ) was f o l l o w e d b y the decrease in i n t e n s i t y at 330 nm (open circles) and wavelength of maximum emission (closed c i r c l e s ) . The e x c i t a t i o n w a v e l e n g t h was 295 nm.  -47-  The  s h i f t  corroborates the  bulk  of  from  the the  to  328  hydrophobic tryptophans  nm  350  nature in  of  the  upon  the  unfolding  environment  native  structure  in  e l l i p t i c i t y  of  of  the  protein. A l t e r n a t i v e l y , increasing thermal  the  7  structure  for  accumulated most  nm,  of  a  by  of is of  t h i s shifted the  of of  as  this the  denaturation  monitoring  cooperative  presence  s t a b i l i z a t i o n  features  as  Since  melting  the  protein  calcium.  -48-  when  1951, the  most  54 to  Linus stable  process  is  of  one  has of  protein  gelsolin  in  the  e l l i p t i c i t i e s  at  210  near  cation,  the  °C,  the  evidence  structure  of  upon  study  transition  divalent to  to  experimental  disruption  thermal  calcium,  temperature  about  The  monitored  CC-helix  molecules,  important  reveals  the  the  was  g e l s o l i n . the  protein  that  denaturation. absence  of  suggested  2  decrease  temperature  s t a b i l i t y  P a u l i n g  the  the  suggesting  temperature  is  4 6 °C.  In  melting that  a  brought  0.8-  o  CD  20  30  40  50  70  T (centigrades) F i g u r e 1 6 . T h e r m a l D e n a t u r a t i o n o f G e l s o l i n F o l l o w e d b y CD m e a s u r e m e n t s . G e l s o l i n u n f o l d i n g (2.6 mg/ml) was followed by the decrease i n e l l i p t i c i t y a t 2 1 0 n m i n 2 0 mM M O P S 1 5 0 mM K C 1 ( p H 7 . 5 ) a n d - 0 . 5 mM C a C l 2 ( c l o s e d c i r c l e s ) or 1 mM E G T A ( o p e n c i r c l e s ) i n a 0 . 1 mm c e l l . T h e f i g u r e s w e r e obtained p l o t t i n g the r a t i o of the e l l i p t i c i t y at temperature T to that obtained at 10 degrees against temperature. P r e c i p i t a t i o n o f g e l s o l i n becomes v i s i b l e at t e m p e r a t u r e s a b o v e 50 °C.  The  p r e c i p i t a t i o n  temperatures this  results,  light the  at  structure). producing  The  215  by  not  was  be  denatured  nm  a  of  by  has  temperature,  with  cooling.  interpretation  the the  at  appears  to  of  the  retain  e l l i p t i c i t y  presence  of  {$-  reputation  of  not  proteins,  in  contrast  to  7 3  hydrochloride  followed  melting  protein  (suggesting  means.  observed  b r o a d minimum i n  denaturation  other  was  reversed  hampers  unfolding  guanidine  g e l s o l i n reported  suggested by  t o t a l  gelsolin  can  thermally  Thermal  denaturation  the  scattering  structure,  centered  of  above  p r e c i p i t a t i o n  Although  some  just  of  the  (Gu-HCl) decrease  induced in  unfolding  e l l i p t i c i t y  and  the HC1  shift  i n e m i s s i o n maximum t h a t o c c u r s  to gelsolin.  denaturation midpoint  The  curves  of gelsolin  obtained  (Figure  upon a d d i n g f o r the  17) h a v e  a  Gu-  Gu-HCl  transition  a t « 1.5 M Gu-HCl. The p r o t e i n seems t o be t o t a l l y  u n f o l d e d a t > 2 M Gu-HCl. The e m i s s i o n maximum o f f r e e t r y p t o p h a n invariant in  w i t h Gu-HCl  emission  c o n c e n t r a t i o n . The c h a n g e s  unfolding i s corroborated  unfolding  observed  maximum may be a t t r i b u t e d t o t h e u n f o l d i n g o f  the p r o t e i n and exposure o f t h e tryptophans The  i s relatively  t o t h e medium.  by t h e c i r c u l a r  dichroism  profile.  1.2358  1.0-  f  * 0.8-  o cz> e£  0.6-  >  —  V  ..  /  •  M  t% a» * *  0.4-  0.2-  i  *>»—o-  0.0-  0  1  2  3  [Gu-HCl]  r  M  4  5  6  F i g u r e 17. U n f o l d i n g o f G e l s o l i n by Gu-HCl. Gelsolin u n f o l d i n g was f o l l o w e d b y : ( c l o s e d c i r c l e s ) s h i f t i n e m i s s i o n maximum upon e x c i t a t i o n a t 295 nm. [ g e l s o l i n ] = 0.035 mg/ml i n 25 mM MOPS - 150 mM KC1 - ImM EDTA a n d d i f f e r e n t amounts o f G u - H C l . (open c i r c l e s ) d e c r e a s e i n e l l i p t i c i t y a t 215 nm. [ g e l s o l i n ] = 1.3 mg/ml i n t h e same buffer. Cell u s e d 0.1 mm. Both curves were o b t a i n e d a t 25 °C. -50-  Comparing chemical  means,  emission  is  of  the  unfolding  it  is  shifted  g e l s o l i n  and  from  from  hydrochloride  induced  explained  the  thermal  i f  are  tryptophans The  the  detected  by  of  proteins  that  the  that  effects  regulation  The  of  the  is  altered  upon  calcium  induced  have  show  some  the  method  been  measurements binding  of  These  one 7  step as  4  on  of  5  fact -51-  7  5  -  7  '  6  This  gelsolin  divalent  cation.  the  gelsolin  methods  '  this  8  6  9  '  7  8  '  7  9  In  c i r c u l a r changes  these  7  regulates  the  in  two  multistep  different  and  that  to  of  changes  5  between  calcium  the  conformational The  characteristics  polymerization.  by  them.  similar  proteins.  structure  binding  preparation  calcium.  the  r e a d i l y  are  process,  some  actin  the  they  opposed  for  investigated  indicate  a  r e l a t i v e l y no  are  introduction,  among  hide  are  Also,  conformational  differences  such  environment.  d i s p l a y  stages.  a  gelsolin  be  after  in  may  g e l s o l i n  detected  that  molecule  upon  in  CD  tryptophans  protein  could  by  the  maximum  guanidine  situation  to  and  unfolding  the  observed  the  methods.  suggests  molecule  of  unfolded,  been  of  thermal in  This  and  in  thermal  environment.  curves  and  mentioned  nm  exposed  aqueous  unfold  has  354  aggregated  different  folded  3 5 0 nm o n  and  intermediate  as  unfolding  real  by  wavelength  structure  c o o p e r a t i v e ,  distinguishable  the  to  t o t a l l y  unfolding  states,  to  328  is  from the  symmetric,  328  residual  not  A l t e r n a t i v e l y ,  that  denaturation.  denaturation  structure  As  found  processes  and  study,  dichroism in  changes  gelsolin were  not  detected  by  experiments without to  some  a  proteins  suggests  gross  are are  that  velocity calcium  conformational  calcium  parvalbumin, changes  sedimentation  and found  binding  upon  activated  change.  where  binding to  effect  -52-  gel  activates  proteins,  calmodulin,  and  This such large  calcium. their  In  f i l t r a t i o n the  is  in  as  molecule contrast troponin,  conformational this  functions.  way  these  Table  III.  Summary  of  Physicochemical  Parameters  horse plasma g e l s o l i n  Parameter Sedimentation  of  Gelsolin.  other  gelsolins  4.9 ,  4.8[4.4]  0.73 -  0.728  a  Coefficient, Partial  s°20,wr  (S)  4.8  Specific  Volume,  a  V(cm /g)  0.727  3  Degree  of  (g Stokes  water/g protein) Radius,R (nm)  Hydration,8  b  b  0.393 4.4 « 3.4 [ 3 . 7 ] « 3.7  3.8  s  a  b  n  Molecular  Weight,M  (kDa):  r  -SDS-PAGE  90  9 1 • 91 « 90 92J n a  b  90^ 93  d  -Gel  f i l t r a t i o n  75  7  -Calculated Mr=6TC TJRsN S ° 0 , w / d " V -Amino a c i d c o m p o s i t i o n  95.3 '  75  p)  2  7  83 , f  f/fo  69  b  81. 6  e  a  1.26[1.38] 1.43  1.2-1.3  b  a  E l l i p t i c i t y , (deg  [6]208 *  10"  -40.4&  - 7 . 8 [-6.1]  3  -8.12[7.0]  cm /dmol) 2  Absorption Coefficient ( m l / m g cm) Melting  temperature,  Transition Gu-HCl  midpoint  Tm  1.4 1.24  1.4  (°C)  by  M  Gu-HCl  (h)  pig  (i)  human  (j)  bovine  (k)  human  plasma.  (a)  rabbit  macrophages.  (b)  bovine  serum.  (C)  human  (d)  rabbit  (e)  pig  (1)  pig  plasma.  1 6  (f)  human  plasma.  6 4  (m)  pig  plasma.  4  (g)  human  plasma.  6 9  (n)  bovine  Brackets:  serum.  plasma.  in  the  5 3  5 2  serum. 6  8 0  5 4  1  presence  of  calcium  -53-  m  45J  46 [54]  1.5  1.8J  1  ions  6 8  plasma.  8  1  serum.  7 9  plasma.  1 5  6  serum.  5 9  h  c  PART  III.  Interaction  As  mentioned  interactions nucleation  with  step  with  Actin.  in actin  of  the can  actin  Introduction, lead to  a c c e l e r a t i o n of  polymerization,  a c t i n f i l a m e n t s and t o c a p p i n g o f a c t i n The  gelsolin  to  severing  polymers.  k i n e t i c s of a c t i n polymerization  can  be  described  which  of  ( c o n s i s t i n g o f a b o u t t h r e e a c t i n monomers),  by a r a p i d e l o n g a t i o n p h a s e and, the  polymer  filaments  length. ' 8  1 1  can The  be  finally,  The  (P)  followed  by  an  average  directionality.  p o s s i b l e t o d i s t i n g u i s h a f a s t g r o w i n g end g r o w i n g end  stable  a steady s t a t e i n  characterized  f i l a m e n t has  (B)  and  It is a  slow  .  nucleation activity  of g e l s o l i n  a b o l i t i o n o f t h e l a g p h a s e and p r o d u c t i o n  i s manifested  that  s h o r t e r and  form f a s t e r t h a n i n samples p o l y m e r i z e d  the  absence  the  nucleating  protein.  by  of filaments  are  of  of  1 2  i n terms o f a l a g phase a s s o c i a t e d w i t h f o r m a t i o n nuclei  the  The  in  capping  and  severing a c t i v i t i e s  of g e l s o l i n r e s u l t i n the  formation  of  shorter  filaments  of  to  already  formed  g r o w i n g end preventing  actin  polymers.  of the  when  gelsolin  is  added  Capping  (binding  to  the  filament) produces s h o r t e r f i l a m e n t s  polymerization  a t t h e f a s t g r o w i n g end.  i s t h o u g h t t o o c c u r by d i r e c t b r e a k i n g The can be  net  fast  of the  Severing  filament.  r e s u l t o f t h e a c t i v i t i e s o f g e l s o l i n on  summarized i n F i g u r e  18. -54-  by  actin  long  >* ^cS>  filament  ^ 65555S$9 * B  s h o r t capped  filaments  F i g u r e 18. S c h e m a t i c R e p r e s e n t a t i o n o f t h e E f f e c t s o f G e l s o l i n on A c t i h P o l y m e r i z a t i o n . A c t i n ( c l e a r c i r c l e s ) . Gelsolin ( d a r k c i r c l e s ) . B, f a s t g r o w i n g e n d . P, s l o w g r o w i n g end.  A. D i f f e r e n c e  The  difference  polymeric plasma  Absorbance.  actin  gelsolin  was on  i n absorbance used  to  actin.  between  study the The  monomeric  e f f e c t s of  difference  and  horse  absorbance  s p e c t r u m o f a c t i n upon p o l y m e r i z a t i o n h a s one p o s i t i v e peak at  280  sample  and  a more p r o n o u n c e d  of globular a c t i n  one  at  232  and a d d i n g s a l t  -55-  nm.  82  Taking  a  at time equal t o  zero time  to  induce  course  increase  in  of  of  actin  19),  actin  difference  Gelsolin (Figure  polymerization,  greatly  it  is  possible  polymerization absorbance  reduces  the  at lag  232  by  to  follow  following  the the  nm.  phase  of  this  demonstrating  the  enhanced  nucleation  polymerization in  the  presence  of  process activity  gelsolin.  F i g u r e 1 9 . N u c l e a t i o n A c t i v i t y o f Horse P l a s m a G e l s o l i n on A c t i n P o l y m e r i z a t i o n . G - a c t i n i n b u f f e r A ( 0 . 5 mg m l in 2 mM t r i s - H C l - 1 mM D T T - 0 . 2 mM C a C l - 0 . 2 mM A T P - p H 7.6) was i n d u c e d t o p o l y m e r i z e b y a d d i t i o n o f M g C l 2 t o 1 . 0 mM. C u r v e 1 s h o w s t h e i n c r e a s e i n a b s o r b a n c e a t 2 3 2 nm o f actin induced t o polymerize i n the absence of g e l s o l i n . Curves 2 and 3 show a more r a p i d development in absorbance in the p r e s e n c e o f g e l s o l i n . T h e r a t i o [ a c t i n ] / [ g e l s o l i n ] = 64 a n d 32 r e s p e c t i v e l y f o r c u r v e s 2 a n d 3 . -  2  -56-  1  B.  Viscometry.  Capillary  viscometry  d i s t r i b u t i o n activity 0.5  mg/ml  CaCl2 were by  -  0.2  actin mM A T P  in  addition  this of  up  molar  when  polymerize actin case  is  pH  shown  7.6  to  1  mM  1.5.  of  IV.  (Table to  to  Table -  Actin  MgCl2  with  a  These  with  A), IV.  consist  1  or  actin i f  it  B) .  The  of  many  filaments.  -57-  a  molar are  when is  steady  it  short,  mM A)  to  state  actin  in  1.2.  A  has  a  to ratio  obtained is  added  f i n a l  0.2  polymerize  v i s c o s i t y  values  -  of  buffer  to  gelsolin  1/50  the  solutions  called  induced  weight  study  mM D T T  reaches  the  to  IV,  (henceforth  Addition of  1.0.  molecular  method  in  reduces  present  (Table  thought  easy  mM t r i s - H C l  solution  filaments is  -  of  viscosity  gelsolin  2  ratio  gelsolin/actin  an  As  in  sensitive  experiment.  viscosity  1/100  relative  offers  gelsolin.  of  used  relative a  of  and  is  to  both  induced  to  preformed  solution  in  each  gelsolin-capped  Sample  rel  viscosity  A  B  F-actin  1.5  1.5  G-actin  1.0  1.0  actin:gelsolin  100:1  1.2  1.2  actin:gelsolin  50:1  1.0  1.0  Table  IV.  Effect  of  Gelsolin  Viscosity  of  Actin  on  the  in  buffer  A;  indicated addition  of  addition  of  the  C.  (A)  molar 1  Actin ratios  mM  MgCl  1 mM M g C l 2  indicated  molar  Interaction  of  on  was an  mixed  and  to  actin  was  gelsolin  complex  in  chelation actin  the  of  is  is  the  Gelsolin  of  with  and  10"  5  M)  gelsolin  to  the  polymerize  by  the  by  the  added  Actin  2  h  later  at  Labelled  with  6-  presence divalent  (Acrylodan).  gelsolin  limiting,  released  x  ratios.  interaction  concentration  (1.18  polymerized  was  Acryloyl-2-dimethylaminonaphthalene  The  Polymerization  Actin with  induced  (B)  2  G-actin  Filaments.  with  produces of  a  2:1  calcium  cation  producing  from a  actin,  when  actin:gelsolin  (Figure the  1:1  actin  1:2  20) . complex,  EGTA  Upon one  resistant  c o m p l e x . 83-86  A  + G  A G 2  -«  AG +  Ca  + A  2 +  Figure 20. Schematic Representation of the Interaction A c t i n and G e l s o l i n , at L i m i t i n g A c t i n C o n c e n t r a t i o n s . actin; (G) gelsolin.  -58-  of (A)  This  model  gelsolins plasma  from  the  suggested result  gelsolin  plasma  C.l  calcium  with  actin  calcium  in  (ADA-actin)  change  position of  and  and  other  binds  a c t i n . is  8  9  the  was  maximal  with  Pope be  actin  et  a l .  lost  as  8  8  a  when  calcium.  interaction  to  pig  activated  investigated Marriot  to  to  of  horse  using  actin  et  a l .  8  9  with  sensitive  acrylodan-labelled  486  nm.  without  e m i s s i o n maximum  cys  374,  the  most  same  site  can  be  be  g e l s o l i n s . and  of  at  decreases  the  The  known  intensity this  of  fluorescence  intensity  Acrylodan  probes  presence  according  is  the  native  are  of  However,  by  that  of  Characteristics.  gelsolin  in  study  plasma  actin  uncorrected  in  A  7  behavior  interact  may  dependence with  8  to  sensitivity  in  the  acrylodan,  Spectral  The  calcium.  the  sources.  reported  present  gelsolin  labelled  been  prepared  with  different  of  the  factors is  The  has  absence  that  of  consistent  several  gelsolin  even, i n  is  wavelength  8  affected 3  '  8  of  probe.  9  5  '  8  7  by  0  -59-  an  (Figure reactive  labelled  large  adding  appreciable 21). cysteine  with  interaction  Thus,  emission  Upon  of  other actin  changes  maximum were  in  expected  F  (a.u.)  420  460  500  540  580  X  (nm)  F i g u r e 2 1 . F l u o r e s c e n c e E m i s s i o n S p e c t r a o f A D A - a c t i n (1 x 10" M) . In the absence (upper spectrum) and presence (lower spectrum) of gelsolin ( 2 x 1 0 ~ M ) . Xex = 390 nm. The m e a s u r e m e n t s w e r e made i n b u f f e r A . a . u . = arbitrary units. 6  6  Quenching complex constant 1.3  M"  value  1  is  is  protected  obtained (Figure  and  quencher  the (see  demonstrate from  for 22).  decreased  (Stern-Volmer (kq)  studies  In to  the 0.909 the  quenched  probe  The by  I"  of  M" .  smaller  The  1  smaller  1.11).  -60-  the  the  in  the  Stern-Volmer  presence  inaccessible  equation  the  quencher.  ADA-actin  constant), more  the  that  is  equal  gelsolin, the  quenching  fluorophore  to  this slope  constant to  the  1.6  M  [KI]  F i g u r e 22. S t e r n - V o l m e r P l o t s f o r t h e Q u e n c h i n g o f Acrylodan i n A c t i n and i n t h e A c r y l o d a n - l a b e l l e d A c t i n g e l s o l i n Complex. Two i d e n t i c a l s a m p l e s o f A D A - a c t i n (0.1 x 10~ M open c i r c l e s ) and two o f A D A - a c t i n + g e l s o l i n i n a m o l a r r a t i o o f 1:1 ( c l o s e d c i r c l e s ) were p r e p a r e d . F v a l u e s were m e a s u r e d f r o m t h e s o l u t i o n s t o w h i c h K I was a d d e d and Fo v a l u e s were m e a s u r e d f r o m t h e s o l u t i o n s t o w h i c h KC1 was a d d e d as a c o n t r o l f o r i o n i c s t r e n g t h and d i l u t i o n . X-ex = 365 nm, Xem = 486 nm. B u f f e r u s e d : b u f f e r A. 7  K I and  KC1  filamentous). experiments, value  at  a c t i n was  a f f e c t the To  avoid  actin  was  state of a c t i n  polymerization used  at  which p o l y m e r i z a t i o n t a k e n t o be  in  the  concentrations occurs.  globular during  s t u d i e s because i t s emission  (globular  The the  s p e c t r u m was  quenching below  state  of  course of not  versus  the ADA-  these  enhanced  and  blue  s h i f t e d as w o u l d have b e e n e x p e c t e d i f p o l y m e r i z a t i o n  had  occurred. The  8 9  quenching  associated shift  of  acrylodan  fluorescence  without  i n the wavelength of maximal e m i s s i o n  -61-  an upon  interaction s t a t i c  of  quenching,  acrylodan  this  in the in  the  the  C.2.  some  static  observed the  to  were  adding the  control  If  the  the not  pursued  this  the  acrylodan-actin interactions  observed  remain i f  observed  measuring  absorption  and  same  behaviour  in  decrease  spectrum  the  in  of  by  to  nm.  buffer  the  samples.  for  sample.  fluorescence The  plot  upon was  two  may  in  the  was  due  shows  -62-  Binding.  o r i g i n adding  used  to  aliquots  obtain  the  the  Titration of  clear  23,  of  gelsolin,  proteins.  small  were  of  the  (Figure  A  Control  another  the  adding  i n t e n s i t i e s  buffer  regarding  observed  binding  actin  390  Stoichiometry  acrylodan-actin  performed  of  for  should  intensity  at  ratio  gelsolin.  question  of  Fluorescence excitation  of  in  in  labelled  absence  gelsolin  of  by of  of  interaction  although  lifetime  the  to  decreases  differentiated  responsible  attributed  quenching.  change  stoichiometry studies  be  lifetime  the  decrease  state  possibilities,  Sensitivity  Despite  that  be  the  absence  Calcium  ground  intensity,  The  and  a  may  and/or  are  emission  presence  two  and  state  i t .  to  could  presence  reflect  to  These  spectrum  ground  gelsolin  g e l s o l i n  thesis,  absorption  and  or  with  fluorescence. in  actin  gelsolin circles).  recorded  at  dilution  was  performed  by  are  presented  as  The  data  values a  in  486  the  continuous  nm  test  increase  on  and in  this mol  ratio  u n t i l  gelsolin  a  plateau  per mol  i s  reached  at  approximately  0.5  actin.  o  0.0  0.2  0.4  0.6  0.8  1.0  gelsolin/actin Figure 23. Titration of ADA-Actin with Gelsolin. The d e c r e a s e i n i n t e n s i t y a t 4 8 7 nm w a s m o n i t o r e d adding gelsolin to actin (F) and t o buffer (Fo) . T h e d a t a is presented as a p l o t o f t h e r a t i o Fo/F a g a i n s t the molar ratio of gelsolin to actin. ADA-actin i n buffer A (open circles). I n b u f f e r A w i t h 2 mM M g C l 2 a n d 5 mM E G T A (closed circles).  When adding  gelsolin,  decrease closed avoid EGTA. actin  ADA-actin  i n  MgCl2  denaturation These  mixed  the decrease  intensity  circles).  only  was  of  results  with  dilution  was p r e s e n t upon  demonstrate  i n the presence  mM E G T A  due t o g e l s o l i n  due t o  actin  5  of  i n these  chelation that  calcium  -63-  effects  the  (Figure  23,  experiments of  to  parallels  calcium  gelsolin ions.  prior  binds  to with to  The  b i n d i n g a n d r e l e a s e o f one a c t i n m o l e c u l e f r o m t h e  A 2 G c o m p l e x was s t u d i e d w i t h p o l a r i z a t i o n a  sample  i s excited  preferential transition  of  excitation. emission.  excitation dipole  direction  with  the  This  polarized  of  light,  the molecules  moments  oriented  electric  vector  photoselection  For a given  measurements. I f  may  there with  parallel of  the  result  i s  their to the  polarized  i n polarized  chromophore, and i n t h e absence o f  energy t r a n s f e r and o t h e r t r i v i a l causes o f d e p o l a r i z a t i o n , the p o l a r i z a t i o n (correlation during  time  viscosity  will  <j>) t h a t  the lifetime  depends  by  value  on t h e s i z e  d e p e n d on r o t a t i o n a l displaces  of the excited and shape  the  fluorophore  the  molecule.  the emission state.  dipole  This  i n turn  of the molecule  and t h e  o f t h e medium. S t u d i e s  polarization  diffusion  on t h e r o t a t i o n a l  motion  measurements r e q u i r e t h a t t h e l i f e t i m e o f be c o m p a r a b l e t o t h e c o r r e l a t i o n  Information  time of  r e l e v a n t t o t h e dynamics o f t h e  m a c r o m o l e c u l e may be d e d u c e d f r o m p o l a r i z a t i o n large proteins with molecular  events. For  weight i n t h e range 1 x 10 , 6  such as immunoglobin M , l i f e t i m e s  of the order  o f 100 n s  a r e a p p r o p r i a t e . T h i s has been found i n t h e case o f pyrene and  some o f i t s d e r i v a t i v e s a s p o l a r i z a t i o n p r o b e s .  proteins  with  correlation  r e q u i r e bound f l u o r o p h o r e s  times  ns, which  gelsolin.  10-150  ns  w i t h s h o r t e r l i f e t i m e s o f up t o  25 n s . The l i f e t i m e o f a c r y l o d a n 4  i n t h e range  Smaller  i s appropriate  i n other  systems i s about  f o r studies  of actin  and  ADA-actin  0.384  upon  in  buffer  excitation  emission  at  reported  by  r i g i d l y  associated  observed  nm.  486  Marriot  a l .  with  This  fluorophore  independent  which  would  be  case  expected  Figure gelsolin has  a  in  is  turn  lowers  to  larger the  affects  and  9  be  of  the  very an  The  to  in  the  the  the  of  0.38  probe  is  depolarization of  the  r o t a t i o n  of  the  of  value  that  tumbling  increase  that  actin of  the  macromolecule,  aqueous  in  The of  buffer  used  polarization  when  actin-gelsolin  actin  diffusion  correlation  <•=  the  of  low.  ADA-actin. than  to  motion  value  observation  suggests  contrast  rotational  the  and  protein.  polarization to  added  8  polarization  similar  global  in  shows  24  volume  volume  the  is  a  nm  the  the  molecule.  in  390  This  is  shows  at  et  r e f l e c t s  A  T|V/kT  =  -65-  time  l/6D  alone.  R  .  The  coefficient  according  complex  to:  larger  and  this  0.42  0.41 -  0.40 -  0,  0.39  0.38  gelsolin/actin  Figure  Gelsolin  that of  24.  Polarization  upon  occurs  calcium  nm a n d (clear  A  circles) of  calcium  adding  against .  is  nm u p o n  dark  reached  there  intensity  however, of of  the  environments  on  of  is  is  decrease  a  added  not  of  increase  in  the  presence  the  two  proteins  excitation  shows  EGTA  free  The  actin  the  approximately  adding  g e l s o l i n .  Fluorescence  polarization  ratio  c i r c l e  to in  at  3 90  effect  of  mol  2  of  actin  chelate  the  polarization.  reach  the  i n i t i a l  actin.  gelsolin  and p o l a r i z a t i o n of  to  and  does  d i s t i n g u i s h between  directly  molar  at  present,  effects  487  5 mM.  ions  The  binding  at  gelsolin  When  value  the  The  the  The  in  Actin.  g e l s o l i n .  decrease,  not  Increase  of  polarization  do  to  monitored  EGTA t o  plateau  mol  This  upon  was  plotted  addition  per  Binding  the  the  two  to  fluorescence actin  s i m i l a r i t y  gelsolin  -66-  ADA-actin  is  of  monomers of  also  the  on  the  acrylodan that  two  suggested  bind  actinby  the  l i n e a r i t y  of  environments  Kurth,  actin  plot. who  3  l a b e l l e d  diazole, The  8  is  not  results of  found  similar  probe  on  plasma it  Coue  responsible present  D.  in  in  upon this  Korn  8  and  in  upon  shown  Other  the  interaction  in  of  Bryan  the  f i r s t  20 its  for  loss  the  of  agreement  of  this  who  8 7  the  that  from  the  preparation or,  with  same  gelsolin.  gelsolin  and  gelsolin.  coworkers  with  that  Figure  during  with  in  and  of  fluorescence  suggest  ions  are  Weeds  s e n s i t i v i t y ,  that  horse  exposure does  the  s e n s i t i v i t y  not  factors are  not  plasma.  Binding  studies  conformational  in  c a l c i u m .  changes  in  5  5  gelsolin  u l t r a v i o l e t  '  7  8  Ultraviolet to  the  changes  binding  using  interaction  calcium  its  from those  fluorescence  thesis  and  5  obtained  as  the  downward  7 - c h l o r o - 4 - n i t r o - b e n z o - 2 - o x a - l , 3-  C i r c u l a r D i c h r o i s m and  Gelsolin  differ  concave  with  actins,  horse  a  d i f f e r e n t  that  calcium  i t s  produce  Two  found  affected  results  p l o t s .  results  enhancement  both  to  abolish  These  and  behaves  has  generally  obtained  those  The  quenching  would  Stern-Volmer and  the  '  7  9  /Absorption Studies  Actin.  literature in  In  of  have  g e l s o l i n this  absorption  measurements.  -67-  which  study,  upon b i n d i n g t o  the  actin  and  been  devoted  result  to  upon  conformational  were  c i r c u l a r  investigated dichroism  Gelsolin and  actin  and  after  the  two  major  in  was  put  in  one  side  the  other.  The  spectrum  inverting  spectra  change  the  (data  The  were  CD  and  the  and  cysteine  actin  and  was  followed  in  shown).  the  curve  then  the  In  x  both  obtained  UV 10"  cases with  the  the  conformational  interaction.  This  of  I  and  a c t i n .  c a l m o d u l i n  conformational  a  9  1  to  changes  Both  is are  model have  a  been  -68-  were  in  mixture  same  either  3.02  contrast  substrates detected.  9 2  the to  of  both was  x  c e l l  10"  5  (data  resembles lack  of  protein  on  proteins to  of  procedure  CD c u r v e  two  analogous in  aromatic  recorded  confirming the  the  and  structure  2 mm p a t h l e n g t h  calculated  a  dichroism.  the  gelsolin,  changes  not  dichroism spectra  The  mixture,  changes  of  region  with  is  gelsolin  secondary  of  25).  b i n d i n g between  conformational  DNAase  UV  spectrum  significant  major  far  M in  of  environment  region 5  the  between  technique.  circular  circular  (Figure  near  5.55  the  the  recorded  actin,  not  in  this  there  c e l l  before  similarity  binding  in  chamber  recorded  that  using  the  The  was  The  by  upon  changes  acids.  double  shows  further  CD r e f l e c t s  amino  and  proteins  and  reports  near-UV  separately  M,  changes  a  mix.  shown),  investigated  far-UV  gelsolin  not  to  upon b i n d i n g d e t e c t e d  Conformational actin  c e l l  of  without  binding  of  the  binding  where  large  240  wav«l«ngth  (nm)  225  $• H H M  I  M  « H  Figure  25.  Conformational  Interaction  gelsolin  mixture The  by  (1)/  A l l  pathlength curve  2:  In A  buffer  [actin]  these  low  is  Similar  stabilization  (2),  were  Curve  = 5.55  a  the  the  10"  in  M.  5  gelsolin  precipitation due of  to  buffers  g e l s o l i n by  =  CD.  e x i s t ,  of  baseline  A  in  x  of  (4).  for  0.05 M  5  in  out.  occurs  ionic  a  IO"  dialysed  carried  low  upon  Spectra  g e l s o l i n : actin  3.02  instability  salts.  -69-  buffer  gelsolin the  G e l s o l i n  calculated  was  were  of  the  UV  one  values  [Gelsolin]  x  reports  to  on  far  experimental  recorded  1:  probably  by  one  measurements  strength of  Changes  detected  and  experiments,  some  i o n i c  as  represents  before  This A.  (3),  spectra  c e l l .  that  buffer.  actin  line  overnight  noted  Actin  volume  broken  mixture.  with  the  mm  and  buffer It in  was this  strength  of  gelsolin  in  suggesting  the  PART  IV.  Interaction  Hydrophobic  of  Horse  Fluorescent  Probe:  naphthalene-6-sulfonate  The  burying  polypeptide structure result, or  in  chain  of  the  crevices  located  in  of  to  detected  the  on  amino the  presence  of  in  removal  of  one  between  gelsolin  was  of  and  of  these and  Ca  actin  on  these  2  ,  9  is  the  As  a  surface  acids  are  or  patches  that  are  patches  have  been  been 4  '  9  found  able  +  -chelators u n i t s .  8  3  to  bind  -  8  7  interactions.  The  the  play in 2  results  noncovalent  the  to  in  Gelsolin,  5  +  gelsolin,  native  % complete. at  a  participate  2  is  the  amino  These  9 3  of  may  have  actin  hydrophobic  patches  found  be  f u n c t i o n . Ca  attain  may  pockets  and  to 100  they  medium.  micromolar  Addition  the  acids  always  If  positions,  proteins  monomers.  hydrophobic  not  acids  protein  amino  solution  protein.  aqueous  native  role  ionic  is  hydrophobic  important  both  aqueous  strategic  formation exposed  of  with  2-(N-Methylanylino)  hydrophobic  protein  hydrophobic  Gelsolin  (MANS).  of in  Plasma  an the  actin  in  the  interaction  and  may  To  involve  investigate  fluorescent  probe  MANS  used. MANS  belongs  derivatives emission aqueous shifted  that  in  a  exhibit  properties. solutions  to  but  solvents  class  extremely  They their of  of  are  anilinonaphthalene environment-sensitive  almost  emission  decreased  -70-  is  nonfluorescent enhanced  polarity.  9  6  -  9  7  and MANS  in  bluehas  been  used  proteins  A.  9  to  including  8  Binding  MANS  of  MANS  26  in  mM M O P S  20  band with  yield with  is  the -  is  buffer  indicative  of  mM K C 1 E).  Its  520 nm.  of  the  in  ImM  the  to  enhanced.  MANS  -  emission  several  420  This  MANS  pH  exhibits  nm  of  and  effect  to  spectrum at  presence  2-propanol.  binding of  EGTA  emission  In  blue-shifted  considerably  solutions  on  9  fluorescence  150  maximum n e a r  spectrum  9  patches  Gelsolin.  shows  called  hydrophobic  a c t i n .  to  Figure  (henceforth  this  characterize  a  7.2  broad  gelsolin,  the  quantum  be  mimicked  can  These  =  of  results  hydrophobic  are  regions  in  gelsolin.  B.  Dissociation  An  adaptation  Scatchard work.  The  disociation  experiments  was  the  and Number  the  Zieler  fluorescence the  This  and  increase  maintained  (Kd)  in at  1  0  was  0  utilized  binding  for  analysis  carried  the  Sites.  protein each  fluorescence  at  gelsolin  -71-  analysis in  data sites  that sites.  A  used  the set  concentration  to  and  between  Several  of  present  (n)  binding  involving 420 nm.  the  was  interaction  assumes  out,  constant  Binding  ligand-binding  of  independent  were  of  t i t r a t i o n  number  constant  gelsolin.  equivalent  of  of  p r o p o s e d by  investigate  and  Constant  the MANS  occurs  in  titration following of (G)  samples while  the  concentration  samples  were  was  MANS  used  concentrations gelsolin  of  was  where  (Mi)  and  increased. MANS  was  while  (M2)  Two  other  kept  the  sets  at  constant  concentration  26.  Gelsolin  x  pH  10  -  M)  6  and  same  Emission in  in  7.2),  in  horse and  (20  in >  t i t r a t i o n  i n i t i a l  slopes  changes  at  determined  E  plasma  wavelength 317  Typical  constant  Spectra  Isopropanol.  buffer  buffer,  Excitation  of  increased.  wavelength Figure  of  of  constant MANS and  the  of  MANS  Emission  mM M O P S  -  gelsolin  150  (2.0  isopropanol. nm.  runs  are  plots  mM K C 1 x  the  in  -  6  MANS ImM  M)  following  ;  Figure  fluorescence  (Smi),  -72-  Bound  of  10"  concentration  concentration, into  Buffer,  Temperature  shown  of  gelsolin  inserted  in  spectra  and  (nm)  in  to  (4.0  EGTA,  the  25  °C.  27.  The  intensity  (Sg),  and  (Sm ), 2  equations:  at  were 1 0 0  (a) K d = (Sm -Smi) / [ (Smi/Mi) - (Sm /M ) ] 2  2  2  (b) n - (Sm/Sg) + (Kd/Sg)[(Sm/M)-Sg/G)]  Where G i s t h e c o n c e n t r a t i o n gelsolin  of gelsolin  w i t h MANS a n d M i a n d M  concentrations  i ntitration of  are the respective  2  i n two d i f f e r e n t  titrations  o f MANS  MANS with  g e l s o l i n . B o t h Smi a n d Sm c a n be u s e d t o c a l c u l a t e n . 2  The in  this  number o f M A N S - g e l s o l i n b i n d i n g manner  after  analysis  sites  calculated  of 5 different  titration  e x p e r i m e n t s was 2.5 ± 0.9 (mean ± s . d . ) . c o n s t a n t was c a l c u l a t e d  The d i s s o c i a t i o n  t o be 0.24 ± 0.13 |iM (mean ± s . d . ) .  MAMS TO WHICH GELSOLM HAS ADDED  GHS0MI TO MHXCH MANS MAS ADDED  [MANS]  [Gttlsolin] (UM)  (UM)  F i g u r e 27. T i t r a t i o n s o f t h e Increments i n Fluorescence I n t e n s i t y a t 420 nm Upon E x c i t a t i o n a t 317 nm. MANS; (A) 6.5 x 1 0 " M a n d (B) 0.65 x 1 0 " M t o w h i c h g e l s o l i n was a d d e d . (C) G e l s o l i n 4.24 x 1 0 " M i n b u f f e r E t o w h i c h s m a l l a l i q u o t s o f a s t o c k s o l u t i o n o f MANS were a d d e d . The i n i t i a l s l o p e s o f t h e p l o t s w e r e u s e d i n (a) a n d (b) t o d e t e r m i n e t h e d i s s o c i a t i o n c o n s t a n t K d a n d t h e number o f b i n d i n g s i t e s n. a.u. « a r b i t r a r y u n i t s . 6  6  6  -73-  C.  Effect  The  of  use  particular to  a  the  of  of  may  extent.  gelsolin  that  (2.4  as  is  (12.0  (1M)  hypothesis  that  gelsolin  a  in  actin  were  shown  that  The  in  of  actin  V).  capable  as  MANS  the  has  one  |1M)  in  that  of  not  presence  alone  alter  of  the  of  the  on  of  severing supports structure  experiments  MANS  it  since  s i t e . "  CD  there  Control  binding  -74-  MANS  affect  viscosity  observation  manner.  MANS  the  of  a  protein  not  region  binding  study  the  does  Moreover,  This  presence  MANS  to  of  suggests  gelsolin  does  probe  peptide  the  (1.2  filaments.  in  of  gelsolin.  gelsolin  Structure.  properties  This  of  significant  run  the  the  (Table  and  fluorescent  presence  effect  structure  JIM)  change  values  demonstrates  actin  extrinsic  significant  secondary  Gelsolin Activity  an  e l l i p t i c i t y  a  on  protein  certain  spectrum not  MANS  has  is the  data MANS Fthe of with been  Relative  Sample  viscosity  [0]2O8 (cleg  Buffer  A  [6]222 cm /dmol) 2  1.0  F-actin  1.5  MANS-actin•  1.5  Gelsolin  1.0  7800  -6100  MANS-gelsolin  1.0  7800  -6100  Gelsolin-actin  1.1  MANS-gelsolin-actin;  1.1  TABLE  V.  Gelsolin. (1.2  |iM)  mM C a C l 2 2  CD  mM w e r e  MANS  1  -  0.2  added  mM  absence  effects  followed  nm  or  by  emission  MANS  by  be  into  and  the  pH  of  A c t i v i t y  7.6).  (7.3  7.5.  in  of  aqueous  as  the  the  changes  trend  (12  KC1  to  150  in  20  requiring JIM)  the  -  Structure  uM),  1 mM D T T  mM a n d  mM M O P S , (1.5  -  MgCl2  polymerized  presence  of  gelsolin 0.2  to  actin.  150  JIM)  mM  and  MANS-gelsolin.  heating  occurs  and  actin  mM t r i s - H C l  samples  Gelsolin  expected the  2.0  (pH  monitoring  maximum  on  in  the  detecting  intensity to  JIM)  Denaturation  be  are  to  MANS.  Thermal  MANS  measurements:  mM A T P  EGTA,  of  The  of  (2.4  measurements.  KC1,  D.  Effect  Viscosity  MANS:gelsolin  fluorescence  in  the  28).  toward  longer  the  gelsolin  environment.  -75-  intensity  wavelength  (Figure  The  complex  at  downward  wavelength unfolds  and  could at  420  which  the  trend  in  emission releases  100  460  410 20  10  30  40  60  50  20  T ( °C ) Figure 28. Thermal Denaturation of MANS:Gelsolin. thermal unfolding of gelsolin (1.0 a n d M A N S (2 |1M) 20 mM M O P S - 150 mM KC1 - 1 mM E G T A ( p H 7.5) was follo by the wavelength o f maximum e m i s s i o n (open c i r c l e s ) fluorescence i n t e n s i t y (closed circles) at 420 E x c i t a t i o n w a s a t 340 nm. a . u . = a r b i t r a r y u n i t s .  The reflect the  fluorescence the  cooperativity  e l l i p t i c i t y  tryptophan indicate p r i o r  properties  at  nm  210  fluorescence that  to  can  but  also  the  interaction  affect  directly of  alter  the  (Figure  changes.  the  only  the  page 47)  processes may  unfolding constant  MANS.  -7 6-  as  page  This  binding  gelsolin with  16,  15,  activated  not  MANS-gelsolin  unfolding  (Figure  thermally  s t r u c t u r a l  temperature  of  of  well  do as or  49) .  The  does the  place  r e f l e c t  that  not  plots  take  of  The in wed and nm.  how  gelsolin, governs  E.  Correlation  The by  tight  the  =  determine  of in  the  plot  and  10  measurements  40  over  unfolding It  of  is  2  temperature,  and  rotational  -  1/3  =  has  p  =  time the  same  MANS the  correlation  ( l / P o - 1 / 3 ) (1+  at  fluorescence  room .  In  temperature, an  in  does  the  range  not  occur  that  the  not  T|,  time,  to  kTT/TTV)  -77-  =  range  that  significant a f f i n i t y  dramatically.  polarization,  fluorescence <|>, i n  were  suggests a  a  dichroism  binding  vary  fluorescence  values  circular  to  with  temperature  from  should  attempt  MANS-gelsolin  temperature  viscosity,  demonstrated  of  0.02)  of  is  polarization  expected  and  T,  samples  Evidence  gelsolin  related  6  the  1/P  the  gelsolin value  samples  °C.  also  gelsolin  Perrin  E  29),  such  to  large  buffer  (Figure  between  between  MANS  correlation  for  extent.  of  MANS-gelsolin  determined  the  MANS-gelsolin.  binding  (MANS  0.3  Perrin  of  s i g n i f i c a n t l y  polarization p  Time  the  lifetime,  folowing  ( l / P - l / 3 ) (1+ 0  P,  to T,  manner:  T/<{»  2.3 i 0  1  .  1  with and  29.  the  emission in  Jt 1 0 *  Plot  The  420  viscosity the  (1  JIM)  nm  to  in  was  Handbook  The Tj/T,  intercept  (l/Pn-1/3),  polarization. this  manner  mobility value  which  The be  is  of  achievable  used  according known:  The  is  MANS of  in  slope  calculate to  the  E  a  while  the  was  interference from the  the  linear  be  use  Plot  solution  of  to  limited  gelsolin  lower  than  glycerol: to the  following  the  given  P  degree  =  intercept rotational relation,  free for  was  the  dye.  water  T/TJ  versus  obtained  of  =  l i m i t i n g in  rotational  indicated  the  |1M)  and  the  value  is  (2  l/P-1/3  estimate  polarization A  nm  Physics.  plot  to  MANS  temperature  from  and  constructed  of  340  values  Chemistry  0.36.  bound  in  wavelength  limiting  somewhat  with  to  can  Po =  MANS  ratio  of  5  MANS:Gelsolin.  buffer  avoid  Kelvin/poise.  • 1  estimated of  1  4  (K-poise" )  for  excitation  «  3  p o l a r i z a t i o n measured  gelsolin  varied. The  Perrin  1  1  2  T/Tt Figure  .  1  by  polarization  this value  0.44. in  a  Perrin  correlation i f  the  plot time  lifetime  can (j) is  T/<|) =  Even than 2  the  ns),  lamp  when  decay  is  the  pulse  the  has  lifetime  4.8  11.1  of  the  correlation  time  ns  of  Calculations shaped  gelsolin  cm /g,  degree  protein),  and,  correlation  the  value the  gelsolin These  20.9  for  p a r t i a l  8  of  determined  for  calculated  are  the  calculation  may  be  not  perfectly of  By  greater  that  calculated f r i c t i o n a l  for  of  a or  r i g i d  1.35-1.40.  -79-  rotational  a  spherically V=  g  gives is  <> j = 37 «  The  shape  1.3  ns.  times  similarity times  for  of  gelsolin.  protein  molecules  errors  associated of  the  correlation sphere  of  times equal  hydrated  These  0.727  water)/(g  correlation  prolate  weighted  calculated.  for  hydration  comparison,  oblate  ratios  of  A  gelsolin  because  to  components  the  be  shape.  longer  bound  volume,  globular  Also,  degree  significant. than  the  spherical.  from  can  a  (<  representative  (0.393  spherical  identical  the  A  80900 Da,  and  confirms  ns.  =  experimental further  MANS decay  specific  hydration,  time  of  time  smaller work  gelsolin,  components.  correlation  is  present  two  gelsolin  mass  buffer  the  assumed  a  not  with  and  for  are  are  with of  contain  be  48 ns a  in with  calculated  values  4-fold  ns  molecular  The  to  lifetime  of  in  fluorescence  can  two  T/TJ  MANS  interacts  found  of  for  available  The  been  of  average  of  width  molecule  lifetimes  3  lifetime  observed.  gelsolin with  though  (slope/intercept)  values  with  protein 3-  to  volume  ellipsoids correspond  to  axial  ratios  between  4  and  7  for  the  oblate  and  prolate  particles. The gelsolin patches also  p o s s i b i l i t y which in  binds  proteins  in  may  actin to the  is MANS,  of  be  having  hydrophobic  complementary  proposed. i t  presence  was of  Due not  MANS.  -80-  to  to the  useful  the  patches  on  hydrophobic  fact  that  to  study  actin both  PART  V:  Covalent  Fluorescence  The  Probe  use  of  fluorescent  1  2  the  higher  that  8  -  3  to  their  the  lower  bands  1  1  1  2  nm)  the  from  yield  which  permit  photophysical certain under  relatively  with  Excimers  are  excited  (M*)  (Scheme  VI).  great  property  dynamic study  studies  and  comes  from  a  relative nm)  systems. long  of  several  i t s  ground  that  a b i l i t y (MM)*  state  are  -81-  (M)  thought  solid the  been  from  going  of  diagnostic solvent. formation  systems  high  quantum  events  can  important  used of  and  advantages  to  the  form  study system  excimers.  form between  aromatic to  4  in  Another  that  1  five  dynamic  be  1  presents  the  and  to  -  polarity  the  of  properties  1  has  a  of  can  0  from  Additional  s e n s i t i v i t y . pyrene  and  2  the  these  lifetime  where  2  1  is  intensities  shows  polarity  polarity  complexes  Excimers  I-V  investigation  structural  transient and  of  (372  1  it  pyrene  numbered  the  local  systems  to  respect, of  extrinsic  partly  structure  The  1 0 9  to  as  m i c e l l e s ,  arises  this  I  permitted  arise  its  use  been  to  of  the  (PIA).  cyclodextrins,  1  spectrum  have  respect  has  2  In  5  microheterogeneous  followed  1  study  vibrational  that  with  and  with  macromolecular  wide  other  be  "  5  emission  (383  micelles  G e l s o l i n  derivatives  the  wavelengths.  III  property  '  4  Their  4  bands  correlation  of  2  of  vibrational  This  1  2  its  study  includes  environment.  observed  the  '  3  and  to  s y s t e m s ,  sensitivity of  pyrene  This  s u r f a c e s .  of  N-(1-Pyrenyl)iodoacetamide,  probes  extensive. polymeric  L a b e l l i n g  occur  an  molecule when  a  sandwiched  arrangement  participating  Scheme  is  molecules.  1  2  possible  between  two  6  VI: k  M*  +  M  M  3  „  *  M+hV  (MM) *  M + M  M + M + hV  monomer emission  The shown  the  potential  in  energy,  Figure (AH),  fluorescence  energy  30.  and  diagram  Because  ground  occurs  at  excimer emission  of  state  longer  for  the  this  reaction  excimer  is  stabilization  repulsion,  (ER) ,  the  wavelengths  than  that  excimer of  the  monomer. In  solution,  greater diffuse  than during  molecule. (Scheme  In VI)  viscosity after  a  a  M  - 4  these  the  intensity of  cases  solvent.  as  a  sum  excimer  enough to  the  a  of  time  -82-  molecules  the  two  a  ground  rate and  depends  monomer  terms.  The  state  on  then rise  K3 the  experiment,  intensity  exponential and  can  constant,  resolved  increases  exponential  concentrations  encounter  controlled In  at  excited  forward  excitation,  i n i t i a l l y  two  forms  lifetime  diffusion  decay  difference  where  their  is  of  pyrene  8  delta-pulse  generally excimer  IO  2  terms. decreases time  of  may The as the  excimer  is  processes  diagnostic are  often  of  excited  referred  to  state  as  reactions.  Birks'  These  kinetics.  ro r  -  Figure 30. Potential Energy Profiles for the Ground and E x c i t e d S t a t e s o f m o l e c u l e s M a n d Q. In t h e c a s e o f e x c i m e r f o r m a t i o n Q = M. The figure shows the energy (E) as a function of the intermolecular separation (r) . AH i s the stabilization energy and  of  energy  the  excimer  respectively. When polymeric enhanced lower  2  the  excimer  chain,  1  2  8  are  ER  is  the  state.  marked  forming  the  permitting  pyrene  local  the  as  with  with  each  repulsion  The hv  pyrene  monomer and  m  hv / e  is  formation often  may  are of  or  occur to  the  excimer  d i f f u s i o n  be  excimer in  by  a  greatly at  much  polymeric  intermolecularly different  intramolecularly  i n  -83-  of  linked  can  formation  attached  other,  are  concentration  Excimer  pyrenes  intermolecular  molecules  observation  molecules  i n t e r a c t i n g  and  ground  7  the  and  excimer  in  fluorescence 1  labelled  interact  medium  the  concentrations.  systems where  of  molecules  same  depends  chains when  chain. greatly  controlled,  the The  on  while  the the  intramolecular  formation  microviscosity  of  structure  chain  the  of  the  formation The  of  study  presence  of  3  4  '  3  observations example, the  on  excimer  solvents monomer ground  had and  state  Ground  excited  precedes case the  of  state  This  pyrene  adsorbed  repulsive  also  forces  3 9  to  in  be  the  counteracted  3  some  unusual  in  from  organic for  spectra  were  state  pyrene  thought  to  t h e i r  subsequent  precede  complex observed  ground  where by  and  same  solutions 7  For  the  covalent in  frozen -  in  3  monomer  ground  been  both  5  3  the  spectrum  state  where  3  -  in  micelle  some  arise  and  also  s u r f a c e s ,  9  studies  are  ground  together  occurs  of  interacting  has  2  excitation  interactions  emission  can  for  characteristics.  species  different  A  to  excitation  interactions  chromophores  led  spectra  same  c r i t i c a l  systems,  Previous  paracyclophanes  states.  the  required  derivatives  t h e i r  solvents,  both  and  fluorescence.  some  two  The  isolated  excimer  its  labelled  the  since  state  of  below  observed.  species.  molecules.  both  conformational  structure  spectroscopic  provided  to  the  some  excitation  excimer  "excimer"  and  aqueous  their  were  attributed  the  in  different  the  on  excimer.  polymeric  and  5  depends  and  preceding  pyrene  in  excimers  medium  surfactants  concentration, p r o t e i n s  the  the  of  of  the  that in  bonds and 1  2  9  the force  excited and  with  bimolecular  rigidity  of  the  environment. Two suggested  hypothetical in  the  case  potential of  pyrene  -84-  energy  curves  adsorbed  to  have s i l i c a  been 3  7  in  order of  to  the  monomer  suggest to  account  the  the  ground upon  the  excimer  forming  the  Pyrene.  of  with  electronic  the  the  repulsion  Hypothetical  R-(P-P)  is  ground  monomer,  unstable  and  from  ground  place,  the  the  state  ground  distance  complex.  3  s t a t e  state  excitation  state.  This of  in  of  to  the  -85-  giving  rise  energy  of  of  complex  the  interaction  the  may  pyrene  molecules  their  ground  state.  Curves  between  Ma,  Da  complex the  transitions  7  differences  the  Potential  radiationless  (b)  s t a b i l i z a t i o n surface.  spectra  respectively.  potential  polarization  respectively.(a)excited  take  the  These  species  intensities the  excitation  31) .  different  in  represents  in  (Figure  two  minimum  counter-balance  31.  differences  excimer  interaction  Figure  the  and  small  state  change and  and  the  presence  monomer  The  for  of  the  and  Ea  and  complex  to  excimer  the  Adsorbed  molecules represent  is  excimer rather  excimer  occurs  may  directly  Similar polymers  in  interactions the  in  that  may  aqueous are  minima  fact  arguments  the  the  this  hold  major  potential  definition,  excited  complexes  dimer  the  which  excited  is  Distinctions the  is  thought  may  term  dimer  in  in  that be  formation.  in  Birks  formed  are  not  excimer  that  to in  also  the  seems  ground  the  kinetics  ground the  however,  are  justified  by  state  do  of  the  that  cysteine  proteins  with  formation  of  environments dichroism protein  in  establish  reacts  apply  specifically  residues pyrene  excimer, and  in  providing  conformational  a  biochemical  the  integrity  and  of  the  fact  of  the  the  is  with  may  some  event,  labelled  -86-  excited from  an  state. made  that  it  complex excimer  pyrene  t h i o l  groups  labelling  on  using  activity it  7  a  result  By  2  cases.  the The  dynamics.  vitro  for  (PIA)  state  always  information  assaying  in  the  not  these  By  Stevens  ground  in  proteins.  derivatives  measurements  ground  configuration not  too.  state  geometry  N-(1-Pyrenyl)iodoacetamide derivative  through  the  certain  important  in  proper  under  distinguish the  for  However,  excimers.  exists  nomenclature, This  is  labelled  responsible  only  medium  dissociated  literature.  not  the  of  hydrophobic  diagram.  occurs  that  molecules  force  energy  interaction  case  where  driving  implies  introduced  the  s o l u t i o n s  conditions,  i n t e r a c t i n g  in  is  protein.  in  of the  cysteine circular of  possible  the to  In PIA  this  were  and  Labelling  EDTA  for  -  of  was  24  pH  protein.  h  the 8  D  ml,  dye.  was  (DTT)  mixture  at was  centrifuge. removed and  by  by  labelled x  g  for  the  same  protein half  20  an  at  and  in  buffer. was  150  further  on  a  a  Bio Prior  c l a r i f i e d  hour.  -87-  by  to  4  1  mM  of  h  excess  the  versus  g  in  any  N-N-  proceed The  bench  top  a  bound  column  4-  rocker.  containing  P2  for  N-(lof  to  mechanical x  of  amount  allowed  D  Rad  of  typically  non-specifically buffer  mM  2 mg/ml,  minimal  15000  1.0  protein  was a  -  groups for  the  with* the  molar  in  mM K C 1  t h i o l  at  fold  reaction  against  f i l t r a t i o n  with  dialysing  presence  competes  temperature  dialysis  uncover  f i r s t  dialysed  dissolved  free  the  gelsolin  a  -  the  then  centrifuged The  gel  versus  room  in  reduce  The  by  mM M O P S  which  with  PIA  D) ,  reduced  dimethylformamide. overnight  with  was  DTT,  pyrenyl)iodoacetamide  to  labelled  PIA.  20  to  protein  mixed  gelsolin  attempt  with  (buffer  The  of  properties.  against:  without  free  an  labelled  7.8  The  in  Gelsolin  d i t h i o t h r e i t o l  buffer  properties  dynamic  Gelsolin protein  the  investigated  structural  A.  study  dye 1  were  mM  DTT  equilibrated  experiment,  centrifugation  at  the  100000  B.  Degree  of  The  Labelling  degree  protein),  was  of  determined  dye  by  simple  344  nm.  The  protein  d e t e r m i n e d .  bands  at  1  3  in the  B r a d f o r d  1  3  curve  prepared  based  in  the  solution from  of  4 65  A  protein  by  degree be  of ±  divalent The  acid amino  in  dye  5 95  nm  the  0.3  was  the  (mol  the  RAD)  dye  is  of  determined  in  by  this  be  This of  assay an  dye  binds  to  with  shift the  unlabelled  solution  at  5 95  concentration  extrapolation. manner  gelsolin)  in  is  acidic A  The  the  calibration  G-250.  the  The  using  blue  protein.  found  not  absorption  instead a  at  been  absorption.  prepared  of  has  of  and  has can  maximum  the  absorbance  PIA)/(mol  dye  gelsolin.  when  law  wavelength  with  b r i l l i a n t  is  concentration  this the  absorption  occurs  protein  at  determined  (BIO  curve  concentration  protein  unlabelled  the  labelling  since  dye)/(mol  Beer-Lambert  of  the  Coomassie  standard  labelled  1.6  known  shift  (mol  concentration  of  assay  plotting  nm a g a i n s t the  gelsolin  as  the  the  absorb  manner  using  to  protein.  same  by  the  of  protein  protein  1  not  wavelengths of  measuring  c o e f f i c i e n t  the  concentration  of  does  The  0  by  defined  application  a b s o r p t i v i t y  determined  PIA-gelsolin.  labelling,  the  the  of  was  the  The  found  absence  to of  cations. primary  but  it  has  composition acid  structure been of  sequence  of  shown  horse  plasma  to  very  gelsolins has  been  -88-  be from  gelsolin similar  other  investigated  is  in  sources  not  amino whose  (RESULTS  AND  DISCUSSION suggest By  that  analogy  horse the of  gelsolin with  the  residues  in  a  ±0.3  present.  implies  that  there  peaks  of  at  and  a  through bands  the  sensitivity lost  6  assumed  that  with  the  of  gelsolin  of  calcium  degree  of  labelled  1  was  '  6  4  of  amount in  this  ions  labelling  selectivity  double  complex 32  to  of  1.6  obtained  and  molecules.  of  with  band  I. the the  shows  nm  excimer.  the Other  that  of  at  The  monomer  three  intensity studies  asymmetric  The  monomer  in  observed of  in  the  the  to the  this  polarity  ratio  of  have  reported  that  and  structure  substitution  -89-  the  structure  not  structure  ring.  emission  4 8 3 nm c o r r e s p o n d s  fine  is  displays  maxima.  to  determination  vibrionic  the  correspond  centered  precludes of  (PIA-gelsolin)  consists  407  pyrene  This  and  and  the  analysis III  and  Figure  broad of  of  derivative.  The  p r o t e i n .  accessibility  increases  absence  desired  gelsolin  nm  emission  emission  the  is  The  labelling  PIA-gelsolin  385  emission  it  sequences  Characteristics.  emission.  spectrum  gelsolin  acid  conserved  5 cysteines.  in  single  amino  gelsolins,  The  9  the  labelled  excimer  highly  selectivity.  that  are  Emission  The  7  performed  higher  a  other  cysteine calcium  reported  is  has  obtain  is  The  gelsolin  was  the  34).  plasma  study  C.  page  fine of  vibrational  the  the  pyrene  o  II  300  350  400  450  500 Wavelength  (nm)  Figure 32. E x c i t a t i o n and Emission Spectra of PIA-gelsolin. (1) Fluorescence emission of P I A - g e l s o l i n at a c o n c e n t r a t i o n 1.0 x 1 0 M i n 2 0 mM M O P S - 1 5 0 mM K C 1 1.0 mM E G T A ( p H 7 . 2 ) . Xex = 3 4 4 n m . ( 2 ) E x c i t a t i o n m o n i t o r e d a t 3 9 6 nm for the monomer a n d (3) e x c i t a t i o n m o n i t o r e d a t 4 8 3 nm f o r t h e e x c i m e r . a . u . • a r b i t r a r y units. - 6  Four  different  spectrum  with  to  suggesting  four,  time. does  It not  was  labelling  experiments  excimer/monomer  found  depend  on  that that the  the the  intensity same  sites  ratio were  excimer/monomer  concentration  -90-  of  yielded  the  always  same close  labelled  intensity  PIA-gelsolin  each ratio  in  the  range is  4.0  well  x  below  excimers the  10"  in  not  conditions  The  the of  the  nm  with  (Figure  excimer  at  The  also  at  at  bands  344 in  of  as  a  the  environment  than  this  The  p o s s i b i l i t y  the  the  p o s s i b i l i t y ,  the  forms  pyrene  results  demonstrate  since  gelsolin  under  page  these  33).  the  at  385 and  in  red  spectrum  of  the  the  excitation on when of  bound pyrene  responsible  t h e i r  properties  the  experiencing  to  aqueous  reveals 332  also  molecules  from  spectrum  at  shift  due  when  shoulder  emission  and  nm  407,  arise,  molecules  the  334  population  state  emission  or  a  can  pyrene  and  at  differences  a  result  PIA-gelsolin  excitation  those  excimer  ground  protein  The  excitation  of  the  in  shoulder  These  two  would  a  nm.  broadening  that  This in  which  excimer  nm a n d  result  different  emission.  the  fluorophore  for  disposition  range  aggregates  than  responsible  proximity.  concentration  excimer  observed  monomer.  spectra  in  of  483  broader  the  interacting  the  higher  molecules  the  of  3 4 6 nm a n d  emission,  protein,  monomer  at  These  4  spectrum  maximum  of  10" ).  or  observed  32).  excitation a  a  are  spectrum  to  is  monomer  values  This  Interactions.  maximum  emission  M.  7  R E S U L T S AND D I S C U S S I O N ,  State  a  (>  dimers  (see  10"  character  excitation  presents  x  concentration  solution  form  Ground  1.0  the  intramolecular  does  D.  -  5  be  enforced s t r u c t u r a l  medium. of  for  suggest may  an  a  To  study  PIA-gelsolin  were  compared  proximate  t h i o l  CH -CH -OH) 2  were  2  and  adducts  pyrenemaleimide),  ground  with  out  with  of  observed  prior as  that  at  while  from  model  compounds  a  to  low  these 33).  t h i o l  emission.  PIA-DTT  interactions  presents that  can  These  in  PIA-ME  presents  excimer be  PIA-ME  33.  Model  to  OH  PIA-DTT  Compounds  PIA-DTT  -92-  were work  and  PIA-ME.  the  .  it  In was  monomer  emission  traced  OH  the  their  I s h i i ,  4  the  in  (PM-tropomyosin) and  3  N-(-l-  analyses  state.  Figure  I s h i i  with  interacting  Lehrer  concentrations  and  (PIA-ME  investigate  connection  tropomyosin of  Lerher  to  excimer  results  purposes  compounds  PM-ME)  in  (SH-  2  molecules  study  ME  (SH-CH -CH(OH)-CH(OH)-  pyrene  model  the  for  and  possessing  Mercaptoethanol,  Figure  (PM-DTT  having  with  emission  PIA  these  pyrene-labelled  agreement  arises  of  state  carried  two  respectively,  the  p o s s i b i l i t y  of  d i t h i o t h r e i t r o l , DTT  labelled  PIA-DTT,  studied  those  functionalities.  and  2  CH -SH)  with  that ground  The to  the  absorption  absorption  380  nm.  the  PIA-ME  of  the  Both  model  suggested  the  studied  in  Table  peak  valley  studies  absorption to the  protein.  The  is  compound  PIA-DTT is  upon b i n d i n g VI)  broader  to .  e x c i t a t i o n  both  By  red  spectrum  of  the  300  in  of  the  PIA-DTT  not  -93-  of  of  the  shown),  the  model of  of  the the PIA  PIA-ME the  to  the  Figure  32.  excimer  is  spectrum the  by  nm,  i d e n t i c a l  spectrum  presents and  spectrum  due  PIA-  in  spectrum  in  dye  the  part  the  the  of  pyrenes  presented  to  of  300-380  is  p,  band.  that  the  great  between  compared (data  the  by  binding  to  spectrum  absorption  PIA-gelsolin  nm  found  spectrum  a  the  systems  given  upon  that  more  the  is  offered  of  qualitative  transition  0-0  absorption  the  excitation  p  altered  PIA-DTT  PIA-gelsolin  absent  is  absorption  region  a  for  of  as  parameter  is  0  to  interaction  manner  The  sometimes  the  resembles  shifted  3  the  suggests  of  of  for  interaction  the  spectra  Below  is  water  .  310  spectrum  state  given  environment  the  to  is  dye  comparison,  In  monomer.  which  in  spectra  and  the  to  VI)  comparable  region  similar  a l . ,  is  that  actin  cases,  i t  the  fact  due  state.  excitation  by  similar  broadening  (Table  of  a  definition  proteins  spectrum  in  and  the  ground  et  ratio  in  is  absorption  (Table  The  intensity  gelsolin  In  VI.  perturbations  ground  PIA-gelsolin  broadening  of  the  the  Herkstroeter  of  In  than  PIA-DTT  measure  PIA-gelsolin  PIA-DTT  suggesting  in  by  of  of  broader  adduct  compound  to  spectrum  are  pyrenes  spectrum  of  the  excitation  a  peak  around  PIA-ME.  This  reflects  285  nm  energy  transfer  from  the  tryptophans  of  the  protein  to  the  pyrene  moieties.  sample  p  (absorption)  p y\.em  — 385  (excitation)  nm  Xem = 4 8 3 nm  PIA-DTT  1.17  1.50  1.12  PIA-gelsolin  1.16  1.5.5  1.11  PIA-ME  1.45  PIA-actin  1.44  TABLE  VI.  p  Spectra of the ratio minimum at  The  fact  spectra  that  the  non-interacting two  for  the  Excitation  and  Absorption  PIA-DTT, PIA-ME, P I A - g e l s o l i n . p i s measured as of the f i r s t a b s o r p t i o n maximum t o the next lower wavelengths.  difference  excitation the  Values  species  in of  the  values  PIA-gelsolin  absorption  pyrenes.  are  p  of and  reflects  the  PIA-DTT,  both  In  the  excitation  resolved  by  selecting  wavelength.  -94-  absorption is  and  due  interacting spectra, the  to and  these  emission  D.  Lifetime  The  time  observing and  at  the  out  at  of  excimer  i n i t i a l  either  Several  the  excimer  or  than of  a  nm,  («  34).  time  2 ns)  decays.  that long  of  is  lived  measured  the  monomer  Excitation  was  observations  can  shorter  the  and both The  the  were  for  significant rise  complex  faster  407  (Figure  measurement  exhibit  one  although  a  nm.  monomer  intensity  f i t  340  functions  the  monomer  excimer component  than  monomer shows  with  a  an very  visible  at  times.  The  could  385  excimer  contribution  longer  at  the  the  decay  response  for  Notably,  resolution  small  emission  nm  made.  and  dependent  485  carried be  Measurements.  be  with long  or  is a  two  lost minor  f i t a  decay  with  short  component  could  not  exponentials.  very  rapidly.  contribution a  be  lifetime  (19.8 (54.3  ns,  of  52  ns,  adequately  Most  The to  f i t  of  decay  total  at  ns.  The  % of  the  total  1.12.  -95-  %  of  the  monomer  long  monomer  70  48  the  excimer  with  times,  emission could  be  intensity)  intensity),  and X  2  =  10«7  •»—r fc> **  t  *? 10  I*.  3  oo  l\  c  ! v II *  c  • •  S102  11  10  e  o z  10*  10° 300  200  100  Time  (nsec)  Figure 34.- PIA-gelsolin E x c i m e r and Monomer Decay Curves Together with Excitation Pulse. B u f f e r u s e d : 2 0 mM M O P S 1 5 0 mM K C 1 -1 mM E G T A ( p H 7 . 2 ) . ( 1 ) D e c a y o f m o n o m e r Xem = 390 nm, (2) Decay of excimer Xem = 4 8 5 n m , ( 3 ) lamp p r o f i l e . Xex = 340 nm.  In M/M,  agreement  the  with  spectral  explained  terms  (Figure  35) .  Single  for  monomer  with  t i g h t l y  ground  state  of  two  are  of  pyrene  labelling of  proteins  while  responsible  excimer.  -96-  for  labelled may  double  molecules  of  1.6  PIA-gelsolin  differently  labelled  emission,  held  degree  characteristics  in  the  the  be  0.3  can  be  species  responsible  labelled  interacting the  ±  emission  proteins in  the  of  the  (a) hv  M  M  +  hv.M  (b)  Figure 35. Model Proposed to Explain the Spectroscopic Properties of PIA-gelsolin. (a) Single labelled molecules are responsible for monomer emission, hVM. (b) Double l a b e l l e d molecules with pyrenes interacting in the ground state are responsible f o r excimer emission, hV£.  With of  this  the  monomer  single  labelled  due  excimer  to  band Under  should the  gelsolin, lifetime  explanation should  excimer of  with  formation. the  conditions  monomer  The  the  presented  the  was  absence  -97-  the  to  response  the  absence  decay  lifetime  emission in  mind,  correspond  proteins  reflect  in  of of  of  any  of  long  the  excited  the  always  in  wavelength dimer.  l a b e l l i n g  present  excimer  PIA  quenching  the  for  of  decay  function  and  could  not  of the be  measured. shown the  The l i f e t i m e  t o vary  protein  dramatically  i n aqueous  them u n s u i t a b l e of  t h e decay  studies that  o f singly  to  lifetimes  a c t i n  1  3  and  2  labelled In  crystallins case,  t h e lifetimes  magnitude measured values  The  occurs  Also,  over  complex t o  the protein  and  PM,  multiexponential bovine  serum  ns)  longer with  proteins  decay  albumin.  f a l l s  when 1  3  5  that base  i s  -98-  t o  o f the o f the  that  o f t h e  model.  single  common  t o  3  PIA-gelsolin  than  The pyrene  3  f o rthe  t h e decay  offered  bound  It  of  1  labelled,  i n t h e range  f o r a  shown  emission.  comparison  The decay  a  environments  been  a  t o  ns f o rPIA  not single  t h e proposed  i s  corresponding  53.8-81.5  were  observed  i n the  o f t h e monomer h a s  o f excimer  time  are expected.  have  and  t h e fact  decay  microheterogeneous by  a  i s complex. The  ns f o r P I A bound  provide  decay.  Other  P I A o r PM showed  i n magnitude  a n d 59.8  (70  o f t h e magnitude  exponentials  the proteins  i s i n agreement  bound  similar  of  makes  i n proteins.  The decay  measured  v a r i a b i l i t y  with  i n t h e absence  times  reported.  excimer,  when  long  This  2  h a s been  a n d absence  derivatives  18.8-27.8,  o f t h e monomer at  monomer  i s  13.5  4.2-5.7,  the latter  but  pyrene  studied.  2.92,  adducts  comparison  a s a sum o f t h r e e of  3  proteins  however,  proteins  1  o f t h e monomer  o f these  reported  f o r  labelled  behaviour,  different been  candidates  model  i n t h e presence  solution.  constant  t h e decay  decay  of the small  fluorophore  phenomenon  as  t o the fluorophore derivatives,  have actin  PIA  c o n s i s t e n t l y 1  interesting  3  0  '  1  3  t o  2  '  1  3  4  and  note  t o that  this  behaviour  these  is  proteins  residue. adducts  observed  contain  However, PIA-ME  solvents.  Also,  multiple  lifetime  ring  due  solution.  and  in  DM F  only  DMF/buffer  buffer,  in  components with  Proton  one  the  amide  conformations different complex  explain  of  that  even  complex  decay  Their these  probes  of  (Table  VII).  The  results  the  probe  two  buffer bond the  observed  of  species  PIA  are  may  have  giving  rise  molecule. and in  could spite  sizes.  -99-  be of  the  that  intrinsic  those  suggest is  or  probes  measured  the  in  DMF  that  in  detected.  In  detected.  The  interaction  syn  species  and  anti  could  have  responsible their  aqueous  S t r u c t u r a l  specific  These  in  an  of  readily  to  small  conformations  were  a  the  suggest  is  behaviour  spectra  cysteine  polyaromatic  results  molecular  both  investigated  1 3 5  d e r i v a t i v e s .  the  that  reactive  NMR  lifetimes  decay  of  fact  N-substituted  PM.  conformation  the  the  co-workers  different  contrast, of  and  pyrene  to  of  highly  exhibit  decay  may  one  several  these  configurations in  of  spite  reported  2  including,  of  heterogeneity  3  PM-ME  exponential  property  1  Scouten  compounds  multiple  only  L i n  and  in  for  relatively  the small  solvent  multiplicity  ppm  DMF  DMF/Buffer  4.1  singlet  8.2  multiplet  10.45  singlet  4.1  and  4.45  singlets  8.2  multiplet  10.4 TABLE  VII.-  NMR  DMF/Buffer  The  profile  the  emission. two  of  the  time  This  the  terms  that  result  the  observed  at  of  485  to  a  rising  excimer of  singlets of  PIA  in  DMF  and  nm  on  the  the  fast  e x c i t a t i o n the  measurement  in  the  excimer  arises  with (<  model  state  positive  proximity.  measurement  reflects  the  excimer  close  very  steady  and  lifetime  the  in  forms  nm  component  that  are  the  483  the  supports  mainly  broadening  f i t  that  evidence  based  at  implies  resolution  was  Data  decay  of  molecules  experimental which  the  absence  pyrene  presumed  Spectral  10.5  E.  pre-exponential indicate  and  2  It  is  respect  to  nsec) .  described  observations spectra.  lifetime  of  from  above  such  The the  This  as  decay excited  complex. The  excimer  magnitude the  region  range not  of  15-35 suffer  each  470-490 °C, major  shows  two  component nm.  where  l i f e t i m e s . is  Thermal CD  has  constant studies  shown  conformational  -100-  The when  in  that  changes,  decay measured  the the  and in  temperature protein  show  that  does both  components increasing  undergo the  with  the fact  does  not depend  a  s i m i l a r  temperature.  that  and  These  the radiative  on temperature  small  results  rate  (Table  Temperature(°C)  Tl(ns)  T  15 20 25 30 35  55.28 54.31 52.98 51.02 50.32  19.96 19.76 19.02 17.86 17.37  are  constant  consistent of  the relative  and  T ,  340  n m a n d X-em = 4 8 5 n m .  2  respectively,  There components two with  contributions  i s  not  B  0.483 0.481 0.480 0.479 0.483  0.517 0.519 0.520 0.521 0.517  a  species  d i f f e r e n t  bichromophoric suggested  that  observed  even  o f t h e measured  the  time  simple  o f the decay.  different  excimers  to  It  may  linked  by  m u l t i e x p o n e n t i a l decay  may b e  f o r  a  formed  situation  may  operate  different  geometries  single with i n are  chain  case  allowed  -101-  2  lifetimes  the  however,  1  of the excimer conformation geometries. of f o r  same  two  but with  1  have  5  m a y be i f  the  Such  PIA-gelsolin the  =  that  spectra  chains  Ti  Xex  Studies  alkyl  different the  the Decay Bi and B  f o r  emission e x i s t .  2  decay.  explanation  similar  l i f e t i m e s compounds  dependent  may b e s u g g e s t e d ,  with  excimers  Bi  Table VIII. Thermally Induced Changes on t h e Parameters o f P I A - G e l s o l i n a t Long Wavelengths. are  upon  VIII).  (ns)  2  decrease  a i f  global  protein-dye behaves  in  configuration. a  similar  solution,  at  present.  This  different  syn-syn, decay  the  four them  to  to  a  be  that  the  may  get  lifetime  of  long  the  responsible  species suggests  It  more  insight was  into  and  the  accessible  the  for  to  short to the  shorter  The  It  1.31  these  long lived  -102-  be  decay  that  some  noted  that  possible of  the  sources  of  components.  potassium  iodide  properties  that  KI  of x  7  decreases  the  M  of as  component. species  the  4.05  results,  quencher  of  plots  10  shown  x _  s  1  -  10  7  1  for  the  the  This  M  in -  1  s  _  1  the  species decay  compared  may  of than  chains  other  the  f i t  less  also  side  component the  good  or  is  with  excimers:  that  Stern-Volmer  of  or  alternatives  occur,  constants  having  syn-syn  different  various  observed  quenching  responsible that  or  of  be  anti-  should  performed  According  for  It  may  equilibrium  suggests  probe, to  anti,  in  an  anti-anti.  the  were  component  and  of  rise  behaves  the  from  actually  components.  provided  short  give  and  as  speculative.  the  more  both  component.  slightly  highly  syn  possible  and  acetamide  molecule  such  exponential  studies  protein.  for  four  around  to  35  to  pyrene  p o s s i b i l i t y  syn-  alternatives  labelled  Figure  The  configurations  Quenching order  PIA. up  is  local  heterogeneity,  in  the  indistinguishable.  explanation  acids  free  result  syn-anti  possible may  may  double  this  amino  in  anti-syn,  the  about  which  rise  the  configurations  present  give  as  bound  conformations,  brings  etc.  probably  of  two  excimer  syn-anti,  could  least  manner  If  to  is the  difference  have  a  local  structure the  in  the  protein  that  makes  i t  more  accessible  to  quencher.  M  [I]  F i g u r e 3 6 . - Q u e n c h i n g S t u d i e s o f t h e Two C o m p o n e n t s o f the Excimer Decay of PIA-Gelsolin. The d e c a y at 4 8 5 nm was measured upon e x c i t a t i o n at 3 4 0 nm w i t h increasing KI concentration. The p l o t is constructed with the r a t i o of the lifetime To i n t h e a b s e n c e o f KI to the lifetime T measured in the presence of the quencher, versus the quencher concentration. Short component: closed c i r c l e s . Long component: open c i r c l e s .  F.  Activity  To the  Structure  investigate  protein  procedure, was  and  caused the  studied.  e l l i p t i c i t y  changes by  the  thermal As  at  of  215  in  the  attached  unfolding  shown nm  PIA-Gelsolin.  in were  of  Figure  secondary label the  the  labelled  37,  measured  -103-  or  structure  as  changes a  of  labelling gelsolin in  function  the of  increasing gelsolin  temperature.  was  found  to  The  occur  favourably  with  the  determined  for  experimental  conditions.  melting at  temperature  4 3 °C  melting  unlabeled  which  PIA-  compares  temperature  g e l s o l i n  of  quite  of  under  46 the  °C same  1.00-  20  30 T  Figure  37.  Thermal  unfolding (closed  in  of  size  was  Precipitation  The gelsolin) shows  that  identical  at  0.1  buffer  215  mm.  becomes  activity was  Stability  in  of  studied the  effects  nm  E  60  upon  visible  the by  the  of  PIA-Gelsolin.  (open  Protein  was  circles)  followed  increasing  at  labelled  The  and  the  =  unlabelled of  thermal  gelsolin decrease  temperature. 1.7  above  mg/ml.  50  gelsolin  viscosimetry.  polymerization  -104-  the  temperatures  capillary and  by  concentration  covalently  labelled on  50  (centigrades)  PIA-gelsolin  circles)  e l l i p t i c i t y  Cell  40  Table  proteins actin.  °C.  (PIAVIII have  These affects  results  the  without  suggest  structure  a  major  of  that  the  gelsolin  effect  on  presence only  i t s  to  of  a  the  label  minor  extent  on  a c t i n  a c t i v i t y  polymerization.  sample  rel  viscosity  F-actin  1.53  G-actin  1.00  Actin  gelsolin  Actin:gelsolin  100:1  1.22  5:1  1.00  Actin:PIAG  100:1  1.23  Actin:PIAG  5:1  1.00  Table  IX.  (1.18  x  mM C a C 1 2 amount It  and  of  was  ratio  -  M)  5  0.2  of  of  The follow  in  mM  to  KC1.  the  buffer  PIAG  buffer  gelsolin  pH  or  A  (2  7.6)  PIAG  time  of  alone.  thermal  changes  PIA-gelsolin  at  in  the  by  as  range  observed  in  range  determined  the  the  the  function  this  study  CD  of  addition  is  molar of  by  the  It  as  the  the  flow  used  to  Figure  38  fluorescence  of  of  temperature. broader  0.2  ratios.  also  PIA-gelsolin. of  -  2 mM M g C 1 2  expressed  were  Actin  appropriate  divided  label  measurements.  -105-  is  intensity of  1 mM D T T  the  indicated  sample  of  a  by  the  viscosity  the  -  mixed with  intensity to  Polymerization.  mM t r i s - H C l  unfolding  relative  compound PIA-DTT  Actin  was  polymerize  Relative  flow  on  emission properties  the  presents  of  mM A T P  induced  150  time  10  Activity  than had  the  model  The  melting  the  melting  been  shown  before  that  temperature page  47) .  their  g e l s o l i n  at  approximately  50  The  aggregation  of  precipitation  formation  in  the  observation  may  that  curve  attributed  excimer  be  at  intensity  intermolecular  due  excimer  protein  to  for  is  °C.  60  with  (RESULTS  excimer  as  suggested  unfolding as  by  r e s i d u a l  broadening  formation  DISCUSSION,  intermolecular  contributions  to  AND  causing  some  The  increasing  molecules  protein,  there  even  °C  allow  unfolded  fluorescence may  p r e c i p i t a t e s  of  of  the  the  excimer  the  from a  melting  decrease  protein  protein  the  and  in to  aggregates.  1.1  •••  1.0  « 8  0.9-  •H ._  a* c  0.8-  •p  0.7-  1*  «  0.6  rH W  10  ->  r  20  T  30  '  40  1  60  50  T (centigrades)  Figure  38.-  Gelsolin. in  buffer  upon with  Temperature  The E  and  increasing the  different  Effect  fluorescence PIA-DTT the  relative above  on  the  fluorescence 50  the  Fluorescence  PIA-gelsolin  temperature.  temperature.  temperatures  in  of  of  Protein °C.  -106-  same The the  at  buffer plot two  were is  10-6  PIAM  followed  constructed  compounds  precipitation  of x  1.0  at  occurs  each at  The  inability  fluorescence been  in  found  studies that  the  the  hydrochloride  the  the  urea  eliminate  with  these  and  1  3  3  a c t i n  1  3  denaturants  decrease  emission. was  a-chymotrypsin  but  The  a l l  such  do  has  Additional have  2  as  shown  guanidine  not  total  excimer  derivative  c r y s t a l l i n s .  tropomyosin  fluorescence  with  the  chemical  excimer  excimer  gelsolin  or  of  of  and  to  labelled  case  cases  temperature  eliminate  temperature  proteins  in  in  of  completely  disappearance  obtained  by  digesting  according  to  Kwiatkowski  of PIAet  a l . " In  spite  observed these  results  do  in  two  protein.  not  produce  functional  derivatives Other  to and  Cooper  of  method drastic  studies  the  by  and  1  et  using his  in  to of  of  of  can ions  proximity and  the  taken  the  PIA-gelsolin,  calcium  gelsolin. be  of  cysteines  2  in  origin  of  close  change  the  in  probe  be and the used  structural  and  Hydrophobic  and  into  account  properties  when  of  pyrene  gelsolin  include  proteins. covalently  a l .  1  3  using  7  of  the  1  3  labelled a  pyrene  maleimide  phosphoinositides interaction  gelsolin  group  the  labelling  should  studied  8  to  spectroscopic  binding 3  up  absence  The  attached  Frieden  gelsolin  the are  the  Janmey  detect  in  interactions  i n t e r p r e t i n g  of  that  cysteines  a  of  characteristics  characteristics  structural  those  complexity  indicate  gelsolin  those  folded  the  spectroscopic  labelled that  of  9  labelled  studied  -107-  to  gelsolin.  between with  gelsolin  adduct  actin  Doi and  fluorescein. labelled  with  lissamine-rodamine investigate studies,  the  of  reported  in  was  reported  pyrene  this  thesis,  maleimide excimer  at  the  nm w a s  emission  in  fact  achieved  was  for  the that 0.2  of  of  course  of  band  spectral This  0.4,  those  excimers  labelled  with  presented  labelled  with  However,  the  maleimide changes  1  4  0  '  1  4  of  lack  Janmey, that  1.3-1.9  to  pyrene  decided  due  with  variable  labelling with  was  of  observed  The  1  use  has of may  the  in  pyrene  corresponding  compared  -108-  these  with  studies  experiments  work.  to  emission  systems.  of  Of  pyrene  gelsolin the  order  c e l l s .  of  additional  degree  in  contrast  also  pyrene  labelling  -  case  observed.  other  the  a l .  in  formation  was  broad  derivative. also  this  the  instead  observed  the  the  time-dependent  maleimide  to  In  the  485  iodoacetamide to  in  chloride  gelsolin et  The  g e l s o l i n  and  of  Janmey  thesis.  maleimide.  this  sulphonyl  location  those  not  B  the been  excimer be  due  authors  obtained  in  CONCLUSIONS  Gelsolin for  the  used  f i r s t  in  this  developed surface  of  is  of  of of  in  Bryan  charge  presence  horse  time  work  by  Treatment  the  from  and  the  divalent  improved takes  ions  cation  isolated  high  yields.  of  and  an  ion  method  the  method  change  to  in  calcium  exchanger  subsequently  produces  purified  The  of  the  upon b i n d i n g  with  and  modification  advantage  protein  plasma  calcium  was  relatively  an  the  plasma  3 0 - 4 0 mg o f  in  the  the  ions.  in  the  absence  gelsolin  per  of  l i t e r  plasma. The  physicochemical  obtained  from  gelsolins sources. protein plasma 280  This  has and  of  refractometry with  those  sedimentation 4.8  S.  Its  specific have  Mr  75  molecular  was  time  and  other  coefficient  of  gelsolin  of  radius  these values  000.  and a  of  was  values  suggests  Comparison  of  from  horse  ml/(mg-cm) of  90  000  at on  by  differential  found  methods. was  to  The  compare i n t r i n s i c  determined to  hydration  this  conserved  e x t i n c t i o n  here  horse  other  The  calculated  with  of  highly  1.4  was  protein  cytoplasmic  Gelsolin  determined  f i r s t  those  weight  by  1  to  is  identity.  obtained  volume =  the  Stokes  Combination  gelsolin  purified  e l e c t r o p h o r e s i s .  gelsolin for  the  plasma  coefficient  apparent gel  of  similar  other  its  absorption  an  are  that  establishes an  coefficient  from  indicates  polyacrylamide  well  plasma  isolated  and  nm  horse  properties  plasma value  be  to  3.8  and  nm.  p a r t i a l  gelsolin  with  the  be  to  value  obtained  by  calculation that  gel of  gelsolin The  is  of well  dichroism  at  a  °C  with  that  the  nucleating  polymerization  increase  in  containing  the  capping  decrease actin  were  two  the  in  used  here  with to  actin  when  the  found  that  gelsolin  the  presence  a c t i n : g e l s o l i n  M.  activity  of  the  half  were  presence  intensity  of and  of  It  was  gelsolin  on  following  an  solutions The  severing  manifested  viscosity  calcium  gelsolin  of  demonstrated.  gelsolin  of  unfolds  nm  that  melting  cation.  by  232  c i r c u l a r  The  plasma  detected  state  by  These  indicate  divalent  at  unfolding  absence  horse  was  in  solutions  the of  gelsolin. polarization  values  of  6-acryloyl-2-dimethylaminonaphthalene study  the  concentration  of  indicate  literature.  curves  in  actin,  the  and  fluorescence,  process.  the  1.5  steady  fluorescence  labelled  of  induced  the  hydrochloride  of  000)  1.2-1.3  confirmed  4 6 °C  proteins,  activity  in  state  absorbance  polymerized The  actin  in  were  ca.  75  intrinsic  further  two  of  the  by  unfolding  a  guanidine of  of  chemically  presence  concentration  ratio  reported  The in  the  (also  determined  then  obtained  data  protein.  and  those  were  in  The  and  globular  unfolds  temperatures  observed  a  studies.  gelsolin  54  f r i c t i o n a l  gelsolin,  measurements  and  its  temperature  profiles compare  f i l t r a t i o n  interacts calcium  complex.  One  interaction of  actin  with  -110-  the  to  gelsolin  limiting.  labelled  ions of  is  of  actin  produce  actin  It only a  molecules  and was in 2:1 is  released  to  divalent  cation.  The  produce  presence  investigated probe  for  in  the  probe  protein. a  It  f i r s t  by  found  on  of  0.24  ± 0.13  these  with  residues residues  of  is  take  the  were  fluorescent  emission  MANS.  maxima  interaction  molecules with  in  of  a  interesting  part  with  can  are  be  the  of  with  the  MANS  bind  dissociation to  speculate  interaction  in  emission  of  the  emission labelled  responsible labelled  The  molecules  is  the  are  the  result  is  indicate  that  state  of  pyrene  of  for  -111-  forced  N-(l-  cysteine  amino  in  the by  the  protein.  The  l i f e t i m e  of  are  at  labelled the  pairs the  acid three  suggested  interaction  the  the  there  Singly  probe two  labelled and  emission  of  That  spectra  with  to  proximity  pyrene  responsible  ground  up  protein  species.  molecules  state  excimer.  for  that  close the  e x c i t a t i o n  covalent  labelled.  of  differently  the  indicated  structure  protein  ground  It  gelsolin  environment-induced  gelsolin  |XM.  studies  labelled  fluorescence  doubly  to  ± 0.9  the  actin.  excimer-like  labelled  2.5  patches  gelsolin  dimensional  and  non-covalent  that  of  sulfonate,  expected  a  pyrenyl)iodoacetamide  the  chelation  using  intensity  manner  Fluorescence  are  time  to  suggested  was  whether  gelsolin  two  upon  patches  florescence  non-covalent  constant on  the  complex  hydrophobic  according  perturbations  in  of  1:1  2-N-(methylanilino)naphthalene  Changes the  a  the least  molecules  monomer,  while  interacting emission of  the  structural  of  in the  pyrene  proximity  in  the  that  protein  involves  covalent  and close  reflects  manner  three  proximity of  incorporation  significant  a  the  of  the  structure  protein.  -112-  the label and  dimensional cysteine does  residues.  not  activity  structure  of  alter the  The in  a  native  EXPERIMENTAL  A.  Preparation  A . l .  Isolation  A . l . a .  Proteins.  of  Gelsolin.  Preparation  Fresh  blood  buffer-dextrose protease  x  of was  inhibitors  g  A.l.b.  Purification  stored  Gelsolin  of  plasma 1  of  1  PMSF  (dissolved  -  to  DEAE-Sephadex NaCl for  2  25 h.  mM  of  The  1  or  in in  To  the a  contained at  1.0  the  mg/1  supernatant 2  1  of  of a  quantities,  was  5 6  for of  changes and  supernatant,  been  2  1  filtered  and  EDTA  a of  began  mg/1  of  methanol),  of  of  25  c e n t r i f uged  at  5  solid  (settled  (pH  35  of  equilibrated  mM C a C l 2  -113-  of  three  by  isolation  gelsolin  presence  7.5)  plasma  the  volume  mixed with  0.5  blood  small  (pH  the  had -  frozen  preparation  CaCl2  that  mixture  the  citrate  needed.  Bryan  against  was  mM T r i s - H C l The  until  plasma,  10 m i n .  A-50  as in  from  method  This  that  1 4 2  a  Gelsolin.  i n i t i a l l y  0.5  3 5 mM.  °C  into  pepstatin  bottled  purified  the  and  collected  20  dialysed  10000 x g f o r added  of  thawed  been  Tris-HCl  -  gelsolin.  with  had  at  was  modification  mM  was  directly  system  leupeptin  centrifugation,  and  that  collected  Plasma  frozen  human  plasma.  anticoagulant  anticoagulant. 5000  of  1  NaCl  volume)  against  7.5) was  and added  was of  5 0 mM stirred to  10  mM  to  the  solution  f i l t r a t e  was  and  applied  DEAE-Sephadex  A-50  mM N a C I  -  A f t e r  extensive  at  gradient.  to  actin  and  0.22  M  lower pooled  saturation), 20  mM M O P S  at  4 °C  on  5-15  the  the  -  150  u n t i l  % polyacrylamide sulfate  A.2.  of  Isolation Actin  skeletal  CaCl2 (pH  was  as -  F-actin  0.2  7.6),  mM A T P at  depolymerizing CaCl2 to  0.2  remove  4  and  against  50  (pH  7.8).  with  the  with for  between  and  of  in  F-  0.15  and  then  by  to  7.8)  by  gels  their  60  dialysis  (pH  checked  0.05-  solutions  (added  mM E D T A  a  %  of  against  and  stored  electrophoresis the  presence  of  2-mercaptoethanol.  Actin. from  according in -  2  an  to  °C.  Before  (pH  acetone  2  -  2  use,  remaining polymers  and or  -114-  -  and 1  i t -  was 1  W a t t ,  -  -  0.01  rabbit 1  4  3  and  0.2 %  mM NaN  dialysed  mM D T T  centrifuged  denatured  of  mM D T T  1 5 0 mM K C 1  mM t r i s - H C l  7.6)  powder  Spudich  mM t r i s - H C l  2 mM M g C l  buffer:  mM A T P  sulfate  was  of  concentrated  gradient  purified  muscle  of  This  column  assayed  (typically  1.0  cm  eluted  were  was  Purity  sodium dodecyl  stored  -  6  column  re-suspension  mM K C 1  x  7.8.  1 mM N a N 3  viscosity  ammonium  used.  -  were  Fractions  by  31  the  proteins  f i n a l  followed  a  to  equilibriated  of  G e l s o l i n  with  to  1 mM E D T A  appropriately  NaCI).  precipitation  -  adjusted  been  washing  0.30  NaCI  pH  ml/h  had  2 5 mM T r i s - H C l  buffer,  a b i l i t y  60  that  equilibration M  i t s  at  -  material.  in  0.2  100000  3  mM x  g  B.  SDS-PAGE.  (Sodium  dodecyl  sulfate  polyacrylamide  gel  electrophoresis).  SDS-PAGE developed in  the  method  by  was  Laemmli.  presence  of  results  constant  gel  to  characterized macro-ions  by  is  ion  smaller  The %  their  its  weight  and  gel  Standards  phosphorylase  b  catalase  (subunit,  dehydrogenase This purity amounts this  (93  as  method any  be  were  The  size  a and  gel  they  larger  to  migrate  If  size,  of  SDS  The  the  a  is  of  the  w i l l  the  be  macro-  obtained  using k i t  from  of  the  i n s t r u c t i o n s values serum  in  parentheses):  albumin  (43  an  000),  (67  000),  and  lactate  of  protein  000). used  as  proteins  detected 5-15  then  only.  was  ovalbumin  also  contaminant  should  purpose  was  separation  c a l i b r a t i o n  the (Mr  This  gel.  a  bovine  36  boiled  with  proteins  the  gelsolin  used:  60 0 0 0 ) ,  (subunit,  pore  the  using  000),  the  size.  the  the  applied.  pore  f o l l o w i n g  manufacturer.  is  general,  of  are  applied  size  in  system  complexes  in  is  that  to  mobility  polyacrylamide  and  chain  average  In  proteins  SDS-protein  assure  buffer  2-mercaptoethanol.  voltage  motion.  molecular  Pharmacia  a  comparable  in  the  an  and  sample  polypeptide  impeded  8  and  the  method,  ratio,  The  mercaptoethanol  according  SDS  length  chains.  using  this  elongated  to  polyacrylamide  In  5 7  out  excess  in  charge  polypeptide  the  carried  in  the  a  present gel.  % polyacrylamide -115-  measure  The  in  considerable gels  gradient  used gels.  for  C.  Amino A c i d  The  amino  according  to  Stein.  The  water 22,  1  4  4  and  4 6,  the at  purified  72  h  and  under dried  values  reported  were  serine,  however,  hydrolysis in  the  cases  described mM M O P S EGTA)  -  and  sectors  of  tray  analysis  with  a  Durrum  The  from  values  by  D-500  obtained threonine  after  72  were  conducted  with  isoleucine.  Experiments.  ultracentrifuge  of  -  E  The (pH  solvent 7.2)  gelsolin  rpm.  -116-  used  and were  double-sector 52000  to  h  analytical  6 5  of  extrapolating  obtained  and  of  NaOH  Most  results  contents  estimated  of  Alberta.  for were  model  12-mm at  a  times.  samples  were  samples  centrifuged  over  average  in  6 N HC1  experiments  mM M g C l 2  a  in  vacuum  of  and  extensively  velocity  Chervenka.  2  105  Moore  The  valine  Velocity  Spinco by  of  dialysed  hydrolyzed  at  performed  °C.  the  The  was  described by  University  were  time.  Sedimentation Beckman  the  hydrolysis  Sedimentation  and  to  was  were  under  submitted  gelsolin  method  vacuum  at  taken  a  samples  analyzer  three  of  gelsolin  acid  zero  D.  conventional  opened,  the  and  analysis  triplicate  pellets, amino  acid  the  and  cooled,  Analysis.  A  0.2  (150  mM C a C l 2  placed  c e l l  mM K C 1  into  regulated  Schlieren  as -  or  20 ImM  separate to  optical  20  °C  system  was  used t o photograph the s e d i m e n t i n g boundary  at 8  min  in  the  intervals.  E. G e l F i l t r a t i o n .  Gel  filtration  studies  l a b o r a t o r y o f P r o f e s s o r S.G. column FPLC  o f Superose system.  globular  6 HR  The  standards  conducted  W i t h e r s (UBC)  10/30  column  were  w i t h a 1 x 30  ( P h a r m a c i a ) on  was  calibrated  (Pharmacia)  that  a  Pharmacia  with  included  cm  a  set  of  (molecular  w e i g h t , Mr a n d S t o k e s ' r a d i u s , Rs, q u o t e d i n p a r e n t h e s e s ) : ribonuclease A 2.09  nm),  (67 000, (232  000,  ovalbumin 3.55  150 mM  KC1  nm) ,  chymotrypsinogen  8.50  (pH 7.0)  nm),  (440  nm).  and 0.5  accessible to the protein  4.81 000,  nm) , 6.10  albumin catalase  nm) ,  S o l v e n t u s e d : 20 mM mM  d e s c r i b e d b y L a u r e n t and K i l l a n d e r , gel  (25 000,  b o v i n e serum  (158 000,  ferritin  (669 000, -  nm),  (43 000, 3.05  nm) , a l d o l a s e  5.22  thyroglobulin -  (13 700, 1.64  C a C l 2 o r 1 mM 6 6  (Kav) was  and MOPS  EGTA. As  the average v a l u e of determined f o r each  protein according t o the r e l a t i o n :  Kav =  (Ve - V o ) / ( V t - Vo)  where: Ve i s t h e e l u t i o n volume o f t h e p r o t e i n o f Vo  i s t h e v o i d volume o f t h e column,  b e d volume o f t h e  column. -117-  and V t  interest,  i s the  total  A  plot  for A  of  the  plot  against  of  (-log a  Kav) / 1  Mr,  provides  the  molecular  against  2  curve  a  Rs  for  for  the  calibration  weight the  of  curve  gelsolin.  standard  protein  determination  of  Rs.  Viscometry.  measurements  thermostatically Cannon-Manning size  was  the  1.0  semi-micro  water  the  performed  bath (size  viscosities protein  at  27  °C  100).  to  a  with  a  The  represent  solution  in  sample  the  that  ratio of  the  alone.  of  Purified -  for  were  viscometer  Relative  time  Labelling  MOPS  controlled  ml.  flow  buffer  G.  of  calibration  V i s c o s i t y  of  log  determination  provides  F.  Kav  Gelsolin  gelsolin  1 5 0 mM K C 1  presence  of  dialysed  for  1.0  4 h versus  typically  excess  of  reaction  was  PIA.  dialysed  mM E D T A  4-8  buffer  ml,  24  7.8  (DTT). E  was  for  - pH  1 mM d i t h i o t h r e i t o l  mg/ml,  dissolved  -  with  mixed  in  a  minimal  was  temperature  allowed on  a  amount to  of  -118-  a  2 0 mM in  was  Gelsolin 20  fold  (Molecular  overnight  rocker.  The  the then at  2  molar  Probes),  N,N-dimethylformamide.  proceed  mechanical  E),  protein  DTT.  with  N-(1-pyrenyl)iodoacetamide  against:  (buffer  The  without  h  at  mixture  The room was  centrifuged free  and  at  15000  against  on  column  P2  buffer.  Prior  clarified  G. l .  the  by  the  of  of  was  dialyzed (pH  7.6)  2.0  mM  for  °C.  removed  of  Actin  the 1  and The by  I. 0 mM D T T .  and  et  h  at  100000 x  of  dye 344  with  2  8  at  gelsolin  protein  of  an  was hour.  PIA-gelsolin, was  2.2  x  determined  (Bio was  determined 10  9  acrylodan  Briefly:  °C.  by  Rad)  .  obtained using M  4  _  a  cm  1  -  1  was  -  the  was  0.2  A and  molar .  1  2  9  dialysing  extensively actin  and  mM C a C l 2 ~ of  to  in  a  the  0.2  same  centrifuged  at  mM to  2  Probes)  for  excess the  actin  2-fold  proceed  the  MgCl  (Molecular  against  was  to  prepared  addition  added  allowed  stopped  according  Freshly  Acrylodan  was  labelled  same  Acrylodan.  reaction  The  the  half  assay  nm o f  formamide  reaction  for  protein  mM t r i s - H C l  4  g  f i l t r a t i o n  versus  labelling  was  with  a l .  gel  by  PIA-Gelsolin.  unlabelled  at  by  The  removed  labelled  protein  bound  were  and  the  labelled  0  centrifuge.  equilibrated  polymerized  dimethyl the  3  top  dye  + 1 mM D T T  of  labelled  against  3  in  bound  degree  with  Marriott  dissolved  the  of  bench  experiment  coefficient  Labelling  excess  a  P2)  Labelling  curve  ATP  4  any  concentration  method  D  Rad  B r a d f o r d  Actin  was  to  concentration  absorption  H.  (Bio  of  calibration the  buffer  determine  using  in  centrifugation  Degree To  g  non-specifically  dialyses a  x  molar 3 h  dye  at was  buffer  +  100000  x  g  3  h  to  labelled and  remove  denatured  protein  s h i f t  was  in  material.  confirmed by  emission  The  the  integrity  increase  maximum  that  in  of  the  intensity  occurs  a f t e r  polymerization.  H. l .  Degree To  of  Labelling  determine  concentration  was  assay  Rad  dye  from Bio  labelling  typically  I.  Absorption  double  were  double was  with  the  actin  10  M"  4  1  cm  used  in  protein  Bradford  protein  as  a  using  at  - 1  the  375  an  nm.  these  standard.  1  4  The  absorption The  5  degree  experiments  was  Spectroscopy.  measurements  Elmer  to  placed  c e l l  to  from was  chamber  recorded  Lambda  in  each  the  the  before  for  and  was  actin after  in  a  actin To  put the  in  added  and  study and  one  other. the  identical  was  gelsolin  inverting  -120-  2  Salt  dilution. of  with  spectrophotometer.  compartment.  Gelsolin and  performed  measurements,  interaction  used.  were  polymerization of  correct  c e l l  4 B  absorbance  induce  other  resulting split  labelling,  determined  actin  difference  sample the  x  the  beam P e r k i n  samples  to  was  absorption  For  of  using unlabeled  1.85 of  degree  0.8.  A l l  one  of  ADA-Act i n .  determined  concentration  coefficient of  the  of  changes  side  cell  buffer  actin,  The to  to  of  a the  spectrum mix.  J.  Fluorescence  J . l .  Spectroscopy.  Steady  State  Steady  state  Perkin  Elmer  Fluorescence fluorescence  LS-5B  spectrometers. measurements excitation spectra  J.2.  (UBC)  A l l  displayed  not  been  and  studies LS-5  samples  wavelength  have  Measurements.  an to  Polarization  measurements  accessory  equipped (Perkin  (Haake).  Results  analyzed  using  calculates  the  with  from  were  fluorescence _.  0.07  f i l t e r  a  at  the  effects.  The  instrumental  carried  out  fluorescence  and  the  PTPOL  for  a  Elmer)  density  software  to  in  the  water  experiments  (Perkin  polarization according  response.  the  same  polarization  circulating  polarization  with  luminescence  in  inner  corrected  Polarization.  instruments  used  avoid  performed  (Columbia)  optical  Fluorescence  were  Elmer)  bath were that  equation:  p=(Iv-GIh)/(Iv+GIh)  Iv  and  Ih a r e  p a r a l l e l polarized for  the  vertically  and  the  polarized  emission  perpendicular,  excitation. unequal  The  with  intensities  respect  correction  transmission  by  -121-  factor,  the  and h o r i z o n t a l l y p o l a r i z e d  to  observed v e r t i c a l l y  G,  accounts  monochromators  light.  of  J.3  Lifetime  Measurements.  Fluorescence the  laboratory  University. London, a  199F  flash II  TN  weighted  by  were  used.  with  2  analysis  Dichroism  Circular  (CD)  visual  equipped  Edinburgh  was  model  performed  employs of  on  a  iterative  the  inspection  were  [8]  = mean  residue  for  f i t of  is the  = 110  = is  the  pathlength  c  = is  the  concentration  used  to  The  a  with  Jasco  photoelastic  control mean  a  and  residue  data molar  from:  Gobs m r w / l O O d c  d  of  with  computer  calculated  weight  e l l i p t i c i t y  w e r e made  equipped  instrument).  [6]=  was  by  Associates,  Measurements.  modified  (Landis  e l l i p t i c i t i e s  water  an  quality  D i c h r o i s m measurements  and  acquisition  The  and  in  Columbia  instrument  which  The  and  spectropolarimeter,  modulator  mrw  analyzer  software  at  Research  Deconvolution  PRA  performed  Turro  counting  deconvolution. X  were  residuals.  Circular  J-20  photon  1710 m u l t i c h a n n e l lamp  N.J.  (Photochemical  single  computer  evaluated  measurements  Professor  PRA  least-squares  K.  of  Ontario)  with  PDP  A  lifetime  in  g/mol  dm in  a  g/cm  3  solution  calibrate  the  -122-  of  D-Pantolactone  instrument  at  220  nm.  in 1  4  6  The  percent  of  a-helix  was  calculated  according  to  Chen  et  al.™ For was  thermal  set  at  the  denaturation indicated  averaged  over  three  allowed  for  thermal  measurement, filtered  a  separate  solutions  gelsolin,  Gu-HCl  solutions  before  any  Nuclear  Columbia Spectra  size  achieved  were  and  were  instrument reading  was  minutes  had  been  P r i o r  to  any  and  f i l t e r  with  were  was  buffer  was  to  a  the  from  Grant  buffers  Millipore. circulating  Resonance  measured,  and  then  shifts  (ppm), used  a  after are  an  stock  indicated  stand  2 h  in  at  Gu-HCl,  solutions the  room  of  legends.  temperature  Measurements.  Bruker  f i r s t  relative as  from  resonance  in  increasing  performed.  iodoacetamide  Chemical  million  the  magnetic  at  prepared  allowed  Magnetic  pyrenyl  which  15  the  centrifuged  measurements  University  formamide, E.  were  Jim p o r e  was  measurement  Nuclear  of  after  the  bath. spectral  L.  and  e q u i l i b r a t i o n .  0.22  For  The  minutes  control  proteins,  wavelength  proteins  through  Temperature water  the  of  in the  with  external  AF a  0.5  250  ml  in  8  of of  NMR  standard.  recorded  at  spectrometer.  containing  2  mg  d6~N,N-dimethy 1 0.02  units,  tetramethyl  -123-  were  sample  addition  reported to  spectra  ml or  silane  of  buffer  parts (TMS  per 8=0)  REFERENCES  1. -  Yin, H.L., 281,  2. -  Kwiatkowski, Cell.  3. -  T.P.  (1979)  Nature  (London)  Mehl,  R.,  and Y i n , H.L.  (1988)  J .  106, 375-384. Kwiatkowski,  (1984)  J .  Yin, H.L., 374a  Biol.  and Cole,  D.J.,  Chem. F.S.  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