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. 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UBC Theses and Dissertations
Isolation and biophysical studies of horse plasma gelsolin Ruiz Silva, Beatriz Eugenia 1991
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