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A fluorescence study of the COOH-terminus region of equine platelet tropomyosin Clark, Ian David 1987

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A FLUORESCENCE STUDY OF THE COOH-TERMINUS REGION OF EQUINE PLATELET TROPOMYOSIN By IAN DAVID CLARK B.Sc. Heriot-Watt University, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1987 ® Ian David Clark In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D E - 6 ( 3 / 8 1 ) ABSTRACT The use o f f l u o r e s c e n t m o l e c u l e s as probes o f p r o t e i n c o n f o r m a t i o n i s r e c o g n i z e d as a t e c h n i q u e which p r o v i d e s v e r y s p e c i f i c i n f o r m a t i o n and has been a p p l i e d , i n r e c e n t y e a r s , to the s t u d y o f the r o l e o f t r o p o m y o s i n (TM) i n the r e g u l a t i o n o f c o n t r a c t i l e p r o c e s s e s . The i s o l a t i o n and s e q u e n c i n g o f TM from h o r s e b l o o d p l a t e l e t s (P-TM) has shown i t t o be d i f f e r e n t from muscle TM, e s p e c i a l l y near the Nh^-and COOH-termini. These d i f f e r e n c e s have been s u g g e s t e d to weaken end-to-end i n t e r a c t i o n o f P-TM m o l e c u l e s . TM's a r e two c h a i n c o i l e d c o i l s and P-TM has c y s t e i n e r e s i d u e s a t the p e n u l t i m a t e COOH-terminus p o s i t i o n o f a d j a c e n t c h a i n s . These can be l a b e l l e d w i t h s u l f h y d r y l - s p e c i f i c f l u o r -e s c e n t p robes t h a t r e f l e c t c o n f o r m a t i o n a l changes i n t h a t r e g i o n o f the m o l e c u l e v i a changes i n t h e i r e m i s s i o n c h a r a c t e r i s t i c s . The r e s u l t s o f experiments on b o t h pyrene (Py) (40) and a c r y l o d a n (AD) l a b e l l e d P-TM show t h a t t h e r e i s a p r e f e r r e d i n t e r a c t i o n o f the COOH-terminus o f P-TM w i t h the N H 2 -terminus o f c a r d i a c TM over t h a t w i t h the N H 2 -terminus o f P-TM. T h i s i n d i c a t e s t h a t the a l t e r e d N H 2 -terminus o f P-TM, w i t h r e s p e c t to muscle TM, i s r e s p o n s i b l e f o r the r e l a t i v e l o s s o f p o l y m e r i z a b i l i t y o f P-TM a t low s a l t c o n c e n t r a t i o n . A d d i t i o n o f a c t i n t o the Py-P-TM (40) and AD-P-TM s p e c i e s showed changes i n e m i s s i o n c h a r a c t e r i s t i c s i n d i c a t i v e o f b i n d i n g to the F - a c t i n f i l a m e n t s , suggest-i n g t h a t the p r e s e n c e o f the probes had n o t a f f e c t e d the f u n c t i o n o f the P-TM a d v e r s e l y . However, the p r e s e n c e o f pyrenes a t the COOH-terminus seemed to reduce f u r t h e r the a b i l i t y o f P-TM to s e l f - p o l y m e r i z e . i i i -Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence p o l a r i z a t i o n suggested that, contrary to the theory of Skolnick and Holtzer on the s t a b i l i t y of two chain c o i l e d c o i l s , the region towards the COOH-terminus i s among the l a s t to lose i t s h e l i c a l character. - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x i ABBREVIATIONS x i i GLOSSARY OF TERMS x i i i A. INTRODUCTION Part 1: Fluorescence Spectroscopy 1.1 Basic Fluorescence 1 1.2 Environmental E f f e c t s 3 1.3 Fluorescence P o l a r i z a t i o n 5 1.4 Excimer Fluorescence 8 Part 2: Actomyosin-Based C o n t r a c t i l e Systems 2.1 Ske l e t a l Muscle . 10 2.2 Regulation of Actin-Myosin In t e r a c t i o n . . . . 11 2.3 Ske l e t a l Tropomyosin 14 2.4 P l a t e l e t Tropomyosin 18 Part 3: Fluorescence and Tropomyosin 23 - V-B. MATERIALS AND METHODS Part 1: Proteins 1.1 P l a t e l e t Preparation 32 1.2 P u r i f i c a t i o n of Proteins 33 1.3 Carboxypeptidase A Treatment of P-TM . . . . 36 1.4 Fluorescent L a b e l l i n g of P-TM 37 1.5 Extent of L a b e l l i n g 38 1.6 S p e c i f i c i t y of L a b e l l i n g 39 Part 2: O p t i c a l Methods 40 C. RESULTS AND DISCUSSION Part 1: Excimer Fluorescence of Pyrene-Labelled P l a t e l e t TM (Py-P-TM) 1.1 Incorporation of Label 43 1.2 D i l u t i o n E f f e c t s 43 1.3 Varying p ' ™ — Keeping [TM] T n^ Constant . . . 45 Py-P-TM i O C 1.4 Varying c ' ™ — Keeping [TM] T n t. Constant . . . 47 Py-P-TM i O C Part 2: Fluorescence of acrylodan-Labelled P l a t e l e t TM (AD-P-TM) 2.1 Incorporation of Label 49 2.2 Emission Properties 50 2.3 Denaturation of AD-P-TM with Guanidine Hydrochloride 51 2.4 Thermal Denaturation of AD-TM Species -P o l a r i z a t i o n Studies 52 2.5 Thermal Denaturation of AD-TM Species and Acrylodan Lifetimes 57 - v i -2.6 Interaction of AD-P-TM with A c t i n 64 2.7 Interaction of AD-P-TM with C-TM 67 2.8 Conclusions 71 BIBLIOGRAPHY 73 - v i i -LIST OF TABLES Table Page 1 Effect of [Py-P-TM] on F 4 8 5 / F 4 0 2 44 2 Lifetime and x 2 values for AD-DTT and AD-TM species at various temperatures 58 - v i i i -LIST OF FIGURES Figure Page 1 Schematic diagram f o r the p r o c e s s e s i n v o l v e d i n e l e c t r o n i c e x c i t a t i o n and d e - e x c i t a t i o n o f f l u o r e s c e n t m o l e c u l e s 2 2 F l u o r e s c e n t m o l e c u l e a c r y l o d a n and i t s p r e c u r s o r , p rodan 4 3 Diagrammatic r e p r e s e n t a t i o n o f e x c i t a t i o n o f a f l u o r e s c e n t m o l e c u l e w i t h v e r t i c a l l y p o l a r i z e d l i g h t and subsequent d e t e c t i o n o f e m i s s i o n a t 90° 6 4 Pyrene d e r i v a t i v e s t h a t r e a c t s p e c i f i c a l l y w i t h -SH groups o f p r o t e i n s 9 5 The main c o n s t i t u e n t s o f s k e l e t a l muscle, a c t i n and myosin 12 6 a) T h i n f i l a m e n t c o n s t i t u e n t s , a c t i n , t r o p o m y o s i n and t r o p o n i n (TN-I, TN-C, and TN-T) 13 b) Model f o r the r e g u l a t i o n o f muscle c o n t r a c t i o n i ) r e l a x e d muscle (pCa =8) i i ) c o n t r a c t i n g muscle (pCa =5) 13 7 End-on view o f the c o i l e d c o i l o f t r o p o m y o s i n l o o k i n g from the NH2-t e r m i n a l end 14 8 Amino a c i d sequence o f a - c h a i n o f tropomyosin. Non-polar r e s i d u e s a r e shown i n s q uares and c i r c l e s 16 9 R e p r e s e n t a t i o n o f Cys-190 c r o s s - l i n k e d QO-TM a t a v a r i e t y o f temperatures, c a l c u l a t e d from a t h e o r e t i c a l model 19 10 a) A l i g n m e n t o f sequences o f S-TM and P-TM to maximize homology 21 ix -b) Relative v i s c o s i t y vs. i o n i c strength r e l a t i o n s h i p f o r s k e l e t a l aa-TM and p l a t e l e t TM 21 11 E f f e c t of increasing amounts of Mg^+ on the binding of s k e l e t a l aa-TM and p l a t e l e t TM to a c t i n 22 12 E f f e c t of a) GuHCl and b) temperature on cros s - l i n k e d and non-cross-linked TM measured by p o l a r i z a t i o n , fluorescence i n t e n s i t y and r e l a t i v e e l l i p t i e i t y 24 13 Schematic model of the p r i n c i p a l TM conformations on the unfolding pathway 27 14 Emission spectra of Py-P-TM ." 29 15 Addition of unlabelled TM's to Py-P-TM and e f f e c t on F ^ g s ^ s s 31 16 P u r i f i c a t i o n of p l a t e l e t TM 34 17 Diagrammatic representation of the fluorimeter used f o r i n t e n s i t y and p o l a r i z a t i o n measurements 41 18 Inte r a c t i o n of unlabelled P-TM with Py-P-TM . . . . 46 19 Inte r a c t i o n of unlabelled C-TM with Py-P-TM . . . . 48 20 Emission spectrum of AD-P-TM 51 21 Change i n r e l a t i v e fluorescence i n t e n s i t y and p o l a r i z a t i o n of AD-P-TM i n low and high s a l t with addition of GuHCl 53 22 P e r r i n p l o t s of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM 54 23 E f f e c t of temperature on AD l i f e t i m e i n AD-C-TM and AD-labelled truncated P-TM 59 24 E f f e c t of temperature on AD l i f e t i m e i n AD-P-TM 60 25 Computer f i t of best exponential decay to the experimental data f o r AD-P-TM at 34.8°C . . . . 61 26 E f f e c t of a c t i n on the emission i n t e n s i t y of AD-P-TM 65 - X -27 Fluorescence quenching by KI of samples of AD-DTT, AD-P-TM and AD-P-TM + a c t i n 66 28 E f f e c t of a c t i n on AD p o l a r i z a t i o n from AD-P-TM 68 29 Interaction of C-TM with AD-P-TM followed by p o l a r i z a t i o n 69 ACKNOWLEDGEMENT I would l i k e to thank Dr. L e s l i e D. Burtnick f o r h i s guidance and encouragement during the course of t h i s project and f o r h e l p f u l com-ments on the thesis i t s e l f . I also wish to thank Beatriz E. Ruiz S i l v a and P a t t i Roy for preparing some of the proteins used i n t h i s work. Thanks also to Dr. L.B. S m i l l i e at the Un i v e r s i t y of Alber t a f o r help i n the i s o l a t i o n of the blood p l a t e l e t s and Dr. Ian Soutar at Heriot-Watt U n i v e r s i t y f o r allowing me access to h i s fluorimeter. - x i i -ABBREVIATIONS A - Anisotropy Abs. — Absorbance AcOH - Ethanoic acid AD - Acrylodan (6-acryloyl-2-dimethylaminonaphthalene) Asn — Asparagine ATP - Adenosine triphosphate Cys — Cysteine Dansyl Chloride - 5-dimethylaminonaphthalene-l-sulphonyl chloride DFP •= Diisopropyl fluorophosphate DMSO - Dimethylsulphoxide DTT «= Dithiothreitol EDTA - Ethylenediamine-tetraacetic acid GuHCl — Guanidine hydrochloride l i e - Isoleucine Leu " Leucine MeOH - Methanol MOPS - 4-(N-morpholino)propanesulphonic acid PMSF - Phenylmethylsulphonyl fluoride Py - Pyrene TLC - Thin layer chromatography TM - Tropomyosin TN - Troponin TPCK - N-Tosyl-L-phenylalanine chloromethyl ketone x i i i -GLOSSARY OF TERMS An enzyme which c l e a v e s amino a c i d r e s i d u e s from the COOH-terminus o f p e p t i d e s o r p r o t e i n s . A t a p a r t i c u l a r pH, a p r o t e i n w i l l have a n e t charge and when p l a c e d i n an e l e c t r i c f i e l d w i l l m i g r a t e towards an e l e c t r o d e w i t h a v e l o c i t y dependent on i t s n e t charge and m o l e c u l a r weight. A form o f c a l c i u m phosphate t h a t i s packed i n t o columns and used to chromatograph p r o t e i n s . P r o t e i n s b i n d v i a i n t e r a c t i o n between n e g a t i v e l y c h a r g e d groups on the p r o t e i n and C a ^ + on the column and are d i f f e r e n t i a l l y e l u t e d w i t h a phos-phate g r a d i e n t . " F r e e z e - d r y i n g " i s c a r r i e d out by f r e e z i n g the p r o t e i n s o l u t i o n i n a d r y i c e / a c e t o n e b a t h and p l a c i n g i t immediately under vacuum. The subsequent v a p o r i z a t i o n o f the s o l v e n t and any v o l a t i l e m a t e r i a l p r e s e n t r e n d e r s the p r o t e i n i n a dry, powdered s t a t e . Sodium d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n v o l v e s the d i s s o c i a t i o n o f p r o t e i n s i n t o t h e i r c o n s t i t u e n t p o l y p e p t i d e s by SDS which b i n d s to the p o l y p e p t i d e s i n such a h i g h b i n d i n g r a t i o as to "swamp" the n a t i v e charge on the p r o t e i n and g i v e an a p p r o x i m a t e l y c o n s t a n t n e t n e g a t i v e charge p e r mass u n i t a l l o w i n g p r o t e i n s to be s e p a r a t e d on the b a s i s o f m o l e c u l a r weight a l o n e . An enzyme which c l e a v e s p r o t e i n s o r p e p t i d e s t o the COOH-terminal s i d e o f b a s i c r e s i d u e s . - 1 -A. INTRODUCTION PART 1. FLUORESCENCE SPECTROSCOPY 1.1 Basic Fluorescence Light emission can reveal properties of b i o l o g i c a l molecules quite d i f f e r e n t from the properties revealed by l i g h t absorption. The process takes place on a much slower timescale, allowing a much wider range of int e r a c t i o n s and perturbations to influence the spectrum. Absorption occurs i n about 10"^ seconds and i s governed by spin s e l e c t i o n r u l e s , which allow only t r a n s i t i o n s between states of l i k e m u l t i p l i c i t y . Since most organic chromophores have an even number of (paired) electrons, the t r a n s i t i o n s are therefore between the ground s i n g l e t state (S Q) and higher excited s i n g l e t states (S]_, S 2 , etc.) (Figure 1). As v i b r a t i o n a l motion i s on the 10"^ second timescale, the absorption process leaves the molecule i n a v i b r a t i o n a l l y excited state (Franck-Condon p r i n c i p l e ) . In s o l u t i o n , t h i s excess v i b r a t i o n a l energy i s d i s s i p a t e d r a p i d l y to the surrounding solvent cage v i a c o l l i s i o n s , quickly r e l a x i n g the molecule to the lowest v i b r a t i o n a l l e v e l of S]_. Further deactivation to S Q can then occur by emission of a photon (fluorescence; rate constant kp) or by competing non-radiative processes including i n t e r n a l conversion, intersystem crossing and quenching of various types (rate constants k^ c, k^ s and kq, r e s p e c t i v e l y ) . Therefore, the f r a c t i o n of excited s i n g l e t s that become de-excited through fluorescence (quantum y i e l d , <£p) i s simply: - 2 -Vibrational relaxation F i g . 1: Schematic diagram f o r the processes i n v o l v e d i n e l e c t r o n i c e x c i t a t i o n and d e - e x c i t a t i o n of f l u o r e s c e n t molecules (1). * F - k F / ( k F + k i c + k i s + kq[Q]) (1.1) where [Q] i s the c o n c e n t r a t i o n o f quencher molecules. The fluor e s c e n c e i n t e n s i t y a t time t , I ( t ) , i s given by: I ( t ) - I ( o ) e - t A F ( 1 2 ) where I ( o ) i s fluorescence i n t e n s i t y a t time zero and the fluorescence l i f e t i m e , r F - ( k F + k i c + k i s + k q f Q ] ) ' 1 . - 3 -1.2 Environmental E f f e c t s Environmental factors can strongly influence the fluorescence emission of a molecule. The emission spectra of most fluorescent molecules i n s o l u t i o n are broad si n g l e peaks lacking i n v i b r a t i o n a l f i n e structure. This i s a r e s u l t of l i n e broadening by solvent interac-t i o n s . The p o l a r i t y of the molecule's environment can s i g n i f i c a n t l y a l t e r the wavelength of maximum emission, fluorescence i n t e n s i t y and l i f e t i m e . Because the excited states of fluorescent molecules tend to be more polar than the ground states, there w i l l be greater r e l a x a t i o n of the excited state i n a polar solvent than i n a non-polar solvent, leading to a decrease i n the S^-SQ energy gap i n the time between absorption and emission. An example of t h i s i s the s h i f t i n emission maximum of prodan (Figure 2) from 392 nm i n cyclohexane to 523 nm i n H2O. Accompanying t h i s red s h i f t i s a decrease i n fluorescence i n t e n s i t y and l i f e t i m e because non-radiative d e a c t i v a t i o n processes are more favored i n polar solvents (k^ c and k^ s increase). In the case of proteins l a b e l l e d with fluorescent probes, informa-t i o n can be y i e l d e d as to the p o l a r i t y of the binding s i t e , and hence conformation of the pr o t e i n i n that region, by observation of changes i n fluorescence emission maxima, i n t e n s i t i e s and l i f e t i m e s upon binding of other proteins, change of temperature, s a l t concentration, etc. Fluorophores possessing a 6-acyl-2-dimethylaminonaphthalene moiety (Figure 2) show fluorescence that i s extremely s e n s i t i v e to solvent p o l a r i t y . Prendergast et a l . (2) synthesized acrylodan from prodan to give a probe that i s reactive s p e c i f i c a l l y with t h i o l groups (e.g. as II C 2H 5 HP-N / H P P R O D A N o II H HP-N H / H P A C R Y L O D A N F i g . 2: F l u o r e s c e n t molecule acrylodan and i t s p r e c u r s o r , prodan ( 2 ) . occur on c y s t e i n e residues i n p r o t e i n s ) , but s t i l l has the s p e c t r a l c h a r a c t e r i s t i c s of the 6-acyl-2-dimethylaminonaphthalene moiety. This makes i t p o t e n t i a l l y v e r y u s e f u l f o r the study of hydrophobic domains and conformational changes i n p r o t e i n s , t h a t c o n t a i n a c c e s s i b l e c y s t e i n e r e s i d u e s . Another proper t y of the f l u o r e s c e n c e emission t h a t i s dependent on the environment of the probe i s the a b i l i t y o f the quantum y i e l d , ^p, to be decreased by a d d i t i o n of dynamic or c o l l i s i o n a l quencher molecules, Q (Equation 1.1). The r a t e of quenching of f l u o r e s c e n c e of a molecule - 5 -bound to a p r o t e i n (kq) w i l l be less than that of the molecule free i n s o l u t i o n and w i l l be r e f l e c t e d i n a change of slope of a p l o t of — vs. [Q] from the Stern-Volmer equation; | a = 1 + k q r o [ 0 j (1.3) where F Q i s fluorescence i n absence of Q, rQ i s the l i f e t i m e of the molecule i n absence of Q and kq i s the bimolecular rate constant f o r quenching. One of the best known c o l l i s i o n a l quenchers i s molecular oxygen, but most p r o t e i n studies are done i n non-degassed aqueous s o l u t i o n where the oxygen quenching e f f e c t i s constant and hence i s not considered. There are many species that are commonly used as quenchers, e.g. Xe, H2O2, acrylamide, N2O, C s + and I", but the mechanisms are not n e c e s s a r i l y the same i n each case. One quencher that we employed i s I". I t i s thought to induce an intersystem crossing to an excited t r i p l e t state, promoted by s p i n - o r b i t coupling of the excited fluorophore and I" (3). 1.3 Fluorescence P o l a r i z a t i o n I f plane p o l a r i z e d l i g h t i s used to excite an i s o t r o p i c fluorescent system, the emission w i l l also be p o l a r i z e d , to a degree dependent on the s i z e , shape and f l e x i b i l i t y of the macromolecule and on the l i f e t i m e of the fluorescent species present. Figure 3 shows a t y p i c a l arrange-ment f o r p o l a r i z a t i o n measurements where a i s the absorption dipole - 6 -Fig. 3: Diagramatic representation of excitation of a fluorescent molecule with verti c a l l y polarized light and subsequent detec-tion of emission at 90° (A). moment of the fluorescent species. The a b i l i t y of u to interact with ve r t i c a l l y polarized light f a l l s off with cos 20, producing a set of excited molecules that is cylindrically symmetrical about the z-axis. Polarization (p) and anisotropy (A) are defined; P " I , , + II (1.4) - 7 -A = + 211 (1.5) where I j j and IX, r e s p e c t i v e l y , are i n t e n s i t y of emission p a r a l l e l and perpendicular to plane of e x c i t a t i o n . The reason for there being two terms for expressing e s s e n t i a l l y the same e f f e c t i s h i s t o r i c a l . The p o l a r i z a t i o n parameter, p, was i n t r o -duced i n the o r i g i n a l l i t e r a t u r e because i t was easy to measure and was based on the d e f i n i t i o n f o r the d i c h r o i c r a t i o , d, used to quantitate Abs AbsX the l i n e a r dichroism of a substance (d = U—: ) . However, due to Abs j | + AbsX the s p h e r i c a l d i s t r i b u t i o n of the emission, the denominator I|| + 2IX i s more correc t as a value that i s d i r e c t l y r e l a t e d to the t o t a l f l u o r e s -cence i n t e n s i t y emitted and hence the term anisotropy was introduced. Anisotropy s i m p l i f i e s many p o l a r i z a t i o n expressions f o r more complicated systems, s p e c i f i c a l l y i n time-dependent studies of p o l a r i z a t i o n decay. For steady-state measurements, which involve an averaging of emission p o l a r i z a t i o n , the term p i s su i t a b l e i n most cases for access to i n f o r -mation on f l e x i b i l i t y , e i t h e r near the binding s i t e s of probes or of a whole macromolecular structure. P e r r i n (5,6) and Levshin (7) derived an equation f o r spheres and small molecules that r e l a t e s the fluorescence parameters, p and r, to r o t a t i o n a l f a c t o r s , v i s c o s i t y (»y) , temperature (T) and molecular volume (V); 1 1 1 1 rkT (_ ) = (_ . ) ( i + ) p 3 Po 3 Vr> (1.6) - 8 -(The V terra becomes more complex for aspherical molecules because rotational diffusion in 3 dimensions must be considered). The term p Q is the intrinsic fluorescence polarization. This is the degree of polarization of the fluorophore i f i t is held r i g i d l y during i t s l i f e -time and i f other extrinsic depolarizing factors are absent. In the case where the absorption and emission dipole moments are parallel (i.e. the angle, fi, between the dipoles is zero), p Q «= 0.5. The general expression relating p D and fi i s ; For most fluorescent molecules, the symmetry of the excited state is the same as that of the ground state and hence fi •= 0 degrees. Polarization measured for these molecules can theoretically have values between 0.5 and 0, depending on the r i g i d i t y and lifetime of the mole-cule. 1.4 Excimer Fluorescence The term excimer is short for excited dimer and is used to describe the species formed between two solute molecules, one in the excited state and one in the ground state; Po = 3cos2g - 1 3 + cos2/3 (1.7) M* + M M + h i / M (1.8) M + M + h i / D - 9 -The most commonly s t u d i e d species t h a t e x h i b i t s excimer formation i s pyrene ( t h i o l s p e c i f i c d e r i v a t i v e s shown i n Figure 4). Pyrene has a r e l a t i v e l y l o n g fluorescence l i f e t i m e which allo w s s u f f i c i e n t time f o r an e x c i t e d molecule t o produce a fa c e - t o - f a c e sandwich arrangement w i t h a ground s t a t e molecule, which i s an a t t r a c t i v e i n t e r a c t i o n . This N-(1-pyrene)maleimide (TM) N-(1-pyrene)iodoacetamide (PIA) F i g . 4: Pyrene d e r i v a t i v e s t h a t r e a c t s p e c i f i c a l l y w i t h -SH groups o f p r o t e i n s . l o v e r s the energy o f D* -» M + M r e l a t i v e to M* -» M and i s the reason t h a t the excimer emission occurs a t a longer wavelength than the c o r r e -sponding monomer emission. The f a c t t h a t the ground s t a t e i s r e p u l s i v e accounts f o r there b e i n g no f i n e s t r u c t u r e i n the excimer emission. For - 10 -pyrene i n ethanol, a pyrene concentration -10~^M i s required for appreciable excimer formation to be detected but i n p r o t e i n studies excimer can be observed at pyrene concentration <10~^M i n cases where the pyrenes are bound i n close proximity to each other. Here the need f o r d i f f u s i o n p r i o r to excimer formation has been removed. This was observed by Betcher-Lange and Lehrer (8) and Ohyashiki et a l . (36) for pyrene-labelled tropomyosin and i n i t i a t e d the use of t h i s , and the other fluorescence p r i n c i p l e s mentioned, for the b i o p h y s i c a l study of tropo-myosin's conformation and i t s importance i n c o n t r a c t i l e systems. PART 2. ACT0MY0SIN-BASED CONTRACTILE SYSTEMS 2.1 S k e l e t a l Muscle A system composed of two proteins, a c t i n and myosin, has evolved i n nature to perform the fundamental process of converting chemical energy into motion. This system i s present i n v i r t u a l l y a l l eukaryotic c e l l s and i s important i n such diverse functions as c e l l d i v i s i o n , p l a t e l e t a c t i v a t i o n , phagocytosis and other c e l l u l a r a c t i v i t i e s i n v o l v i n g mechan-i c a l s t r e s s , t o r s i o n and t r a n s l o c a t i o n . The knowledge that has been obtained concerning the structure and function of a c t i n and myosin, and the proteins associated with them, comes l a r g e l y from studies on ske-l e t a l muscle. Sk e l e t a l muscle i s a highly s p e c i a l i z e d tissue designed to produce movement and force i n a s p e c i f i c d i r e c t i o n and for t h i s purpose i t - 11 -contains large amounts of a c t i n and myosin organized into c l o s e l y packed, hi g h l y ordered arrays (Figure 5). In mammalian s k e l e t a l muscle, myosin i s aggregated to form t h i c k filaments, while the t h i n filaments, which are anchored to perpendicular l i n e s of p r o t e i n c a l l e d a - a c t i n i n ( Z - l i n e s ) , contain a c t i n as well as the regulatory proteins troponin and tropomyosin. The molecular mechanism f o r t h i s i n t e r a c t i o n i s not yet f u l l y understood but the generally accepted model for contraction i s that f i r s t proposed by Huxley (9) on the basis of e l e c t r o n microscopic and x-ray d i f f r a c t i o n studies. This s l i d i n g filament model has the myosin heads c y c l i c a l l y forming crossbridges with, and r e l e a s i n g the a c t i n to p u l l adjacent Z-lines c l o s e r together, shortening the muscle (Figure 5). 2.2 Regulation of Actin-Myosin Interaction The i n t e r a c t i o n of a c t i n and myosin i s c o n t r o l l e d , i n s k e l e t a l muscle, by two proteins, troponin (TN) and tropomyosin (TM). Both proteins are attached to the t h i n filament and, depending on the [ C a 2 + ] , e i t h e r allow or prevent myosin heads from binding to a c t i n . A c t i n filaments are made up of i n d i v i d u a l a c t i n monomers, G-actin, that have polymerized to filamentous or F-actin. In the t h i n filament, the r a t i o of proteins i s 7 G-actin:1 troponin:1 tropomyosin (Figure 6a). Troponin has three subunits TN-I, TN-C, and TN-T, which i n h i b i t myosin binding, bind calcium and bind tropomyosin, r e s p e c t i v e l y . Potter and Gergely (12) produced a model for the calcium-dependence of muscle - 12 -S'H Z A I s'band line band band -14-i Z sarcomere Z / \ / \ Myofibril A band 2 nun ium mm IIIIII mm mm nun j inn / lamer asin) " H IIIIII i min 11W \ z mm t in in i HUH i min I m i n i IIIIII Thick filaments (myosin) IIIIII mm IIIIII IIIIII IIIIII nun MINI IIIIII 7 Thin filaments (actin) \ / Cross sections at points indicated F i g . 5: The main c o n s t i t u e n t s of s k e l e t a l muscle, a c t i n and myosin (10) - 13 -TN-C Actin Tropomyosin Thin f i l a m e n t c o n s t i t u e n t s , a c t i n , tropomyosin and tr o p o n i n . (TN-I, TN-C, and TN-T) (11). Model f o r the r e g u l a t i o n of muscle c o n t r a c t i o n i ) r e l a x e d muscle (pCa «8), i i ) c o n t r a c t i n g muscle (pCa =*5) (12). - 14 -contraction (Figure 6b). Upon addition of Ca^ + and subsequent binding to TN-C, a conformational change occurs i n which the tropomyosin s l i d e s deeper into the grooves of the a c t i n double h e l i x and TN-I moves away from the myosin binding s i t e , allowing myosin access to a c t i n . 2.3 Skeletal Tropomyosin As depicted i n Figure 6a, tropomyosin i s a r o d - l i k e p r o t e i n 42 nm long. I t has a molecular weight of 66,000 and i s composed of two highly h e l i c a l subunits (>90%) wrapped around each other to form a c o i l e d c o i l , s t a b i l i z e d by hydrophobic i n t e r a c t i o n s between the chains. Since two turns of an a - h e l i x contain seven amino acids, a two stranded a - h e l i c a l c o i l e d c o i l can be s t a b i l i z e d by i n t e r a c t i o n s between hydrophobic residues at p o s i t i o n s 2 and 5 (Figure 7). The sequence of tropomyosin can be set out so that i t f i t s t h i s c r i t e r i o n v i r t u a l l y along i t s f u l l length (Figure 8). A high proportion of a c i d i c and basic residues are found i n p o s i t i o n s 6 and 1, respectively, allowing further s t a b i l i z a t i o n of the c o i l e d c o i l v i a e l e c t r o s t a t i c i n t e r a c t i o n between p o s i t i o n 1 on one chain and p o s i t i o n 6 on the other. Rabbit s k e l e t a l muscle contains two types of polypeptide chain, a and /3, which are present i n an a/f} r a t i o of approximately 4/1. The a and forms have an i d e n t i c a l molecular length of 284 amino acids, but show 39 d i f f e r e n c e s i n t h e i r amino a c i d sequences and migrate d i f f e r e n t l y on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Tropomyosin was f i r s t i s o l a t e d i n 1946 (15) from r a b b i t s k e l e t a l F i g . 7: End on view of the c o i l e d c o i l of tropomyosin looking from the NH 2-terminal end (13). muscle and i s very stable and r e s i l i e n t to extremes of pH, high temper-atures (up to 85°C) and addition of organic solvents. In muscle, TM binds h e a d - t o - t a i l along the t h i n filament (Figure 6a). I t i s t h i s h e a d - t o - t a i l i n t e r a c t i o n that i s responsible for i t s polymerization at low s a l t concentration (10 mM T r i s - H C l , pH 8), as observed by increased v i s c o s i t y (16), and for i t s cooperative binding to a c t i n (17,18,19). The polymerization involves overlap of 8 to 9 residues of the C00H-and NH2-terminii of adjacent TM molecules. When residues 274-284 were removed from the COOH-terminus of r a b b i t cardiac TM (19), the a b i l i t i e s to polymerize and to bind to F - a c t i n were removed, i l l u s t r a t i n g the importance of the h e a d - t o - t a i l i n t e r a c t i o n i n TM for t h i n filament assembly and regulation. The actual binding s i t e s to a c t i n are not - 16 -1 2 AcMet -Asp 14 ' 15 16 Asp - Lys -Glu 28 29 30 Asp - Lys -Lys 42 43 44 Glu - Leu -Val 56 57 58 Glu Leu -Asp 70 71 72 Lys Leu -Glu 84 85 86 Asp Val -Ala 98 99 100 Glu Leu -Asp 112 113 114 Lys • Leu -Glu 126 127 128 Gly • Met -Lys 140 141 142 Lys • Met -Glu 154 155 156 H e • Ala -Glu 168 169 170 Lys • Leu -Val 182 183 184 Arg • Ala -Glu 196 197 198 Glu • Leu -Lys 210 211 212 Gin • Ala -Glu 224 225 226 Glu - H e -Lytf-238 239 240 Arg - A l a -Glu 252 253 254 Ser - H e -Asp 266 267 268 Lys - Tyr -Lys 280 281 282 Asp - Met -Thr 3 f A^ 5 A l a AIle/-Lys 17 / l 8 \ 19 Asn •\Ala/-Leu 31 /^32\ 33 Ala AAla/-Glu 45 /^4ty\ 47 Ser \Leu/-Gln 87 r& Ser "VLeu. 6 7 Lys -Lys 20 21 Asp -Arg 34 35 Asp -Arg 48 49 Lys -Lys 62 63 Glu - A l a 76 77 Lys -Lys 90 91 Arg -Arg 104 105 Glu -Arg 118 119 Lys - A l a 132 133 Ser -Arg 146 147 l i e -Gin 160 161 Arg -Lys 174 175 Ser -Asp 188 189 Gly -Lys 202 203 Asn -Asn 216 217 Gin -Lys 230 231 Asp -Lys 244 245 Arg -Ser 258 259 Asp -Glu 272 273 Glu -Glu 8 9 10 - Met -Gin - Met 22 23 24 - Ala -Glu - Glu 36 37 38 - Ser -Lys - Gin 50 51 52 - Leu -Lys - Gly 64 65 66 - Leu -Lys - Asp 78 79 80 - Ala -Thr - Asp 92 93 94 - H e -Gin - Leu 106 107 108 - Leu -Ala - Thr 120 121 122 - Ala -Asp - Glu 134 135 136 - Ala -Gin - Lys 148 149 150 - Leu -Lys - Glu 162 163 164 - Tyr -Glu - Glu 176 177 178 - Leu -Glu - Arg 190 191 192 - Cys -Ala - Glu 204 205 206 - Leu -Lys - Ser 218 219 220 - Glu -Asp - Lys 232 233 234 - Leu -Lys - Glu 246 247 248 - Val -Thr - Lys 260 261 262 - Leu -Tyr " A l a 274 275 276 - Leu -Asp - His 208 Glu F i g . 8: Amino polar acid sequence of a-chain of tropomyosin. Idealized non-residue positions are shown in squares and circles (14). - 17 -known but along the length of the TM molecule there i s a pseudo-repeat of 14 regions, each having 20 residues. There i s a zone of 8 non-polar amino acids i n each region which i s implicated i n the actin-binding of TM along i t s length (20). End-to-end in t e r a c t i o n s of TM molecules are reduced i n the presence of s a l t s (KC1, NaCl), suggesting that these i n t e r a c t i o n s may be polar or i o n i c i n nature. But, as a large percentage of the h e l i x - h e l i x interac-t i o n i s hydrophobic, the presence of s a l t increases the s t a b i l i t y of the i n d i v i d u a l c o i l e d c o i l s . This i s borne out by thermal denaturation studies (21, 22) which involve following the temperature-dependent loss of a-helix by c i r c u l a r dichroism, which measures % h e l i x i n TM. The r e s u l t s show that the main loss of h e l i x i n cardiac TM (C-TM) i s around 50°C i n the presence of 0.5 M NaCl and at 38°C i n 4 mM NaCl. In high s a l t , both C-TM and S-TM show a small loss of h e l i x at approximately 30°C, known as the p r e t r a n s i t i o n . This has been assigned to s e l e c t i v e i n s t a b i l i t y i n the region of the molecule containing Cys-190 from evidence by fluorescence (23) (see Part 3), c i r c u l a r dichroism (23), NMR (24), ESR (25), and d i f f e r e n t i a l scanning calorimetry (26). Because TM i s almost e n t i r e l y a - h e l i c a l , i t has been used to provide experimental r e s u l t s to te s t the theories of Skolnick and Holtzer concerning a-helix to random c o i l t r a n s i t i o n s of two chain c o i l e d c o i l s (27,28). Using values f o r h e l i x i n i t i a t i o n and propagation f o r each amino a c i d i n the sequence and for i n t e r - h e l i x i n t e r a c t i o n free energy, they computed t h e o r e t i c a l % h e l i x vs. temperature p l o t s for native aa-TM and for aa-TM cros s - l i n k e d at Cys-190. Their r e s u l t s agreed well with the experimental r e s u l t s c o l l e c t e d using c i r c u l a r - 18 -dichroism. The large p r e t r a n s i t i o n present i n c r o s s - l i n k e d TM was proposed by Lehrer and coworkers to be due to the f a c t that i n the undisturbed native double h e l i x , the two s u l f h y d r y l s of the Cys 190's, which are both i n i n t e r i o r p o s i t i o n 2 (Figure 7), cannot quite reach to form a d i s u l f i d e bond. Hence, the presence of the d i s u l f i d e introduces a considerable s t e r i c s t r a i n i n the h e l i x and leads to l o c a l i z e d unfold-ing of the h e l i x i n the Cys-190 region (23), r e s u l t i n g i n a p i c t u r e of the molecule at about 35°C that i s double h e l i c a l at both ends and a pinched "bubble" of random c o i l centered at the c r o s s l i n k (21,29). However, the model of Skolnick and Holtzer suggests that the presence of a bubble of random c o i l i s not possible due to the inherent i n s t a b i l i t y of such a conformation. Their h e l i x p r o b a b i l i t y p r o f i l e s show that during the p r e t r a n s i t i o n , instead of forming a bubble, the molecule unfolds e s s e n t i a l l y from the C00H-terminus to the c r o s s l i n k (Figure 9). The issue i s s t i l l not completely resolved and further experimentation i s being done to resolve the problem, including our own work (presented i n t h i s t h e s i s ) , which u t i l i z e s the fluorescence of acrylodan bound to a cysteine group second from the CO0H-terminus of p l a t e l e t TM to follow the e f f e c t s of temperature on that region of the molecule v i a f l u o r e s -cence p o l a r i z a t i o n from measurements. 2.4 P l a t e l e t Tropomyosin I t has been known for over two decades that c o n t r a c t i l e proteins e x i s t i n non-muscle c e l l s and are responsible for many processes i n v o l -- 19 -F i g . 9: Representation of Cys-190 cross-linked aa-TM cal c u l a t e d from t h e o r e t i c a l model, with a s t r a i n extending 7 residues to the NH2-terminal side and 21 residues to the COOH-terminal side of the c r o s s - l i n k . S o l i d l i n e s , >75% h e l i x , dotted l i n e s , 25-75% h e l i x ; random c o i l s , <25% h e l i x (28). v i n g movement, such as c e l l m o t i l i t y , c e l l d i v i s i o n , phagocytosis and c l o t r e t r a c t i o n . A c t i n i s the major c o n t r a c t i l e p r o t e i n present i n non-muscle c e l l s (as opposed to myosin i n s k e l e t a l muscle) and i s found i n i t s monomeric form (G-actin) at much higher percentages than i n muscle. In processes such as c e l l motion where the c e l l shape i s changing, there - 20 -i s a constant reordering of the a c t i n filaments (F-actin) v i a rapid interconversion between F- and G-actin which i s regulated by a v a r i e t y of proteins i n c l u d i n g tropomyosin (30). The f i r s t non-muscle tropomyosin to be i s o l a t e d was that from human p l a t e l e t s (33) , recognizable as a tropomyosin from i t s >90% h e l i c a l content, p r e c i p i t a t i o n at pH 4.6 and i t s s i m i l a r amino a c i d composition to that of s k e l e t a l TM. The main feature that distinguishes i t and most other non-muscle TM's from s k e l e t a l TM i s t h e i r smaller s i z e , the sub-unit molecular weights generally being 26-30,000 daltons. To date, the only f u l l y sequenced non-muscle TM i s equine p l a t e l e t TM (P-TM) and i t contains 247 amino acids i n each subunit, with cysteines at p o s i t i o n s 153 and 246 (34). As the r a t i o of 0/a i n P-TM i s greater than 2:1, the amino a c i d sequence obtained i s most l i k e l y that of the fi chain. From an analysis of t h i s sequence, some important conclusions concerning the structure of P-TM and i t s r e l a t i o n s h i p to muscle TM can be drawn. F i r s t l y , i t i s c l e a r that P-TM and muscle TM are c l o s e l y related, with a high degree of homology i n t h e i r sequences extending from r e s i -dues 81 to 260 of S-TM (44 to 223 of P-TM). In t h i s region, no inser-tions or deletions occur, but immediately to the C00H- or NH2-terminus sides of t h i s s ection a sudden change takes place and the sequences of the two proteins s t a r t to d i f f e r greatly. In a l i g n i n g the sequences of the muscle and p l a t e l e t TM's to maximise homology (Figure 10.a), i t can be seen that the lower molecular weight of p l a t e l e t TM i s accounted for by two 21-residue deletions within the f i r s t 80 residues of the muscle TM. I t should also be noted that the NH2-terminus of P-TM i s extended by 5 residues. Although these a l t e r a t i o n s do not seem to disrupt the - 21 -a-helical structure of the protein, they profoundly affect P-TM's ab i l i t y to function as does S-TM in vitro. The head-to-tail polymeriza-SKELETAL TM PLATELET TM 1 6 1 22 44 S9 81 , 258 2S4 VA I •;••.<••••.•:•:.•.•• :• -:-'7Xm 1 6 27 28 43 44 221 247 F i g . 10 a) Alignment of sequences of S-TM and P-TM t o maximize homology. Speckled r e g i o n - sequences very s i m i l a r ; c r o s s -hatched r e g i o n - sequences d i f f e r e n t ; blank r e g i o n - areas of S-TM which are deleted i n P-TM (11). K C I ( M | Fig.10: b) R e l a t i v e v i s c o s i t y vs. i o n i c s t r e n g t h r e l a t i o n s h i p f o r s k e l e t a l aa-TM (•) and p l a t e l e t TM (O) (31). - 22 -tion of P-TM at low ionic strength is much less favored than that of S-TM as can be seen from the viscosity data (Figure 10.b). The binding of actin to P-TM, as measured by a sedimentation assay, requires [Mg2+] = 6-8 mM, as opposed to 2 mM for S-TM, for stoichiomet-r i c binding (Figure 11) and this, along with the fact that there is a higher temperature in vivo (37°C vs. 20°C i n binding studies) and free cytoplasmic [Mg 2 +] is less than 1 mM, suggests that P-TM would bind poorly to free actin filaments in vivo. However, recent studies on pig platelet TM (35) have shown that binding to actin is >90% complete in MgCI2 (mM) Fig. 11: Effect of increasing amounts of Mg^+ on the binding of skeletal aa-TM (•) and equine platelet TM (•,A) to actin. Assays were performed with skeletal actin (•,•) and platelet actin (A) (32). - 23 -ph y s i o l o g i c a l s a l t conditions (100 mM KC1, 1-2 mM Mg 2 +), suggesting that there could be a s i g n i f i c a n t f u n c t i o n a l difference between P-TM's from d i f f e r e n t animal species. PART 3. FLUORESCENCE AND TROPOMYOSIN U t i l i z i n g the r e a c t i v i t y of -SH groups on the cysteine residues at p o s i t i o n 190 on the a-chain and 36 and 190 on the /J-chain of TM, Ohyashiki et a l . (36) l a b e l l e d the s i t e s with fluorescent probes N-(l-anilinonaphthyl-4)maleimide (ANM) and N-(l-pyrene)maleimide (PRM). They showed that as the degree of polymerization of ANM-TM was reduced by the ad d i t i o n of KC1 up to 3 M, the emission maximum was blue s h i f t e d and the fluorescence p o l a r i z a t i o n increased, suggesting that i n mono-meric TM the -SH groups are buried deeper i n the hydrophobic core between the h e l i c e s than i n polymerized TM. The same e f f e c t s were observed upon binding of ANM-TM to F-actin, i n d i c a t i n g that the a c t i n may be inducing the same conformational change i n the polymerized TM that occurs upon depolymerization of the TM. In the reconstituted t h i n filament, ANM-TM + F-ac t i n + troponin, p o l a r i z a t i o n i s 0.42 i n the absence of C a 2 + and drops to 0.39 i n the presence of C a 2 + , which approaches the value f o r ANM-TM + F-ac t i n alone (0.36). The explanation put forward was that troponin strengthens the binding between TM and F-a c t i n and the addit i o n of Ca reverses the e f f e c t . There are 6 Tyr residues per a-chain of TM and t h i s f a c t was u t i l i z e d by Lehrer (23) to study the e f f e c t on TM structure caused by - 24 -oxidation of the -SH groups on Cys-190 to form S-S bonds, c r o s s - l i n k i n g the adjacent chains. Tyrosine has a low quantum y i e l d but s u f f i c i e n t fluorescence was detected to allow i n t e n s i t y and p o l a r i z a t i o n studies on the i n t r i n s i c fluorescence of TM. I t was found that both cross-linked and non-cross-linked TM showed responses i n fluorescence p o l a r i z a t i o n to guanidine hydrochloride-induced unfolding and temperature-induced unfolding that were very s i m i l a r to those observed by c i r c u l a r dichroism measurements of h e l i x loss (Figure 12). The non-cross-linked TM (dashed l i n e s ) show major h e l i x loss at lower [GuHCl] and lower temperature than c r o s s - l i n k e d TM, although the c r o s s - l i n k e d species does show a pretran-s i t i o n that Lehrer assigned to p a r t i a l unfolding i n the Cys-190 region. The p o l a r i z a t i o n losses r e f l e c t increased f l e x i b i l i t y of the parts of the molecule containing Tyr side chains whereas the fluorescence inten-s i t y increases with increased [GuHCl] are a r e s u l t of reduced quenching from neighboring carboxylate groups as the chains d i s s o c i a t e . The reason that the fluorescence i n t e n s i t y decreases with temperature for both TM species f a s t e r than for L-Tyr i n s o l u t i o n i s that the quenching e f f i c i e n c y of the carboxylate groups increases with temperature. The f a c t that the two chains of TM are capable of being cross-l i n k e d at Cys-190 made them p o t e n t i a l l y able to produce excimer f l u o r -escence i f s u l f h y d r y l - s p e c i f i c pyrene d e r i v a t i v e s were attached to Cys-190 residues on neighbouring chains. This was demonstrated by Betcher-Lange and Lehrer (8) who worked with r a b b i t s k e l e t a l a and /S chains that had been i s o l a t e d by ion-exchange chromatography i n 8 M urea. Subsequently, they renatured aa-TM s p e c i f i c a l l y , to remove the p o s s i b i l i t y of l a b e l l i n g the y3-chain at Cys 36. They showed that the - 25 -a) b) Ttmperolurt PC) F i g . 12: E f f e c t o f a) guanidine h y d r o c h l o r i d e (GuHCl) and b) tempera-t u r e on c r o s s - l i n k e d TM (—) and n o n - c r o s s - l i n k e d TM ( ). Top, change i n p o l a r i z a t i o n ( p ) ; Middle, change i n f l u o r e s -cence i n t e n s i t y ( F ) ; Bottom, change i n r e l a t i v e e l l i p t i c i t y (m/[*]0> 222 nm (23). l o s s o f excimer w i t h i n c r e a s e d [GuHCl] preceded the l o s s of h e l i x which suggested t h a t the conformational change t h a t i s being probed by the - 26 -excimer fluorescence must be a chain separation i n the v i c i n i t y of Cys-190. They could produce s p e c t r o s c o p i c a l l y d i f f e r e n t products by l a b e l l i n g with N-(l-pyrene)maleimide (PM) at pH 6.0 and pH 7.5 as a r e s u l t of a secondary r e a c t i o n of the succinimido-ring with OH" at pH 7.5 (22). The cleavage of the r i n g by OH" allows r o t a t i o n about two a d d i t i o n a l s i n g l e bonds between the pyrene and the S-atom and r e s u l t s i n a greater degree of excimer formation than f o r the sample l a b e l l e d at pH 6.0. The presence of appreciable excimer below the p r e t r a n s i t i o n temperature suggests that, since pyrenes cannot stack around the outside of the molecule (37), pyrene-pyrene hydrophobic i n t e r a c t i o n s can open the chain around Cys-190, thus c r e a t i n g an equilibrium between chain-open and chain-closed conformations i n the native p r o t e i n . This i s supported by evidence from l a b e l l i n g with a maleimide d e r i v a t i v e of a non-hydrophobic n i t r o x i d e spin probe (25) which showed very l i t t l e p erturbation of the h e l i x unfolding p r o f i l e . Lehrer also showed that the i n t r o d u c t i o n of pyrene moieties on the Cys-190's of TM strongly i n h i b i t s low s a l t polymerization. This i s s u r p r i s i n g considering the COOH-terminus i s 94 residues away and suggests that the pyrenes cause a conformational change that can be transmitted to the end of the mole-cule. Also, the e f f e c t of s a l t on excimer i n t e n s i t y i s very s i m i l a r to i t s e f f e c t on depolymerization and may then be c o r r e l a t e d with a s a l t -induced conformational change near Cys-190. However, i n a l a t e r study (22), I s h i i and Lehrer show the loss of v i s c o s i t y to be p r i m a r i l y due to the 10% l o s s of h e l i x caused by l a b e l l i n g at Cys-190. I s h i i and Lehrer (38) proposed an equilibrium scheme (Figure 13) that explains the presence of both monomer and excimer i n the emission spectra of pyrene-- 27 -N D M 'mimiiiinttiti II i / Fig. 13: Schematic model which assumes two probe conformations M and E, in the principal TM conformations on the unfolding path-way. N is the native, chain-closed, f u l l y h e l i c a l state observed at low temperature; X is the chain-open, partially unfolded intermediate; D Is the f u l l y unfolded, dissociated state (38). TM and how excimer fluorescence increases by means of a temperature-dependent shift in equilibrium from chain closed state (N) to chain open state (X) associated with the pretransition. They showed that the presence of the label causes a decrease in TM binding to F-actin, possibly the result of reduced end-to-end interactions. When pyrene-TM was bound to F-actin, excimer fluorescence did not increase with temper-ature, indicating that F-actin stabilizes TM by inhibiting the N -» X transition. Further stabilization of TM was also observed with binding - 28 -of myosin heads (SI) to the F-actin/pyrene-TM complex, even at low [ S l ] / [ a c t i n ] r a t i o s ( i . e . <l/7), i n d i c a t i n g that myosin could produce long range e f f e c t s on the state (or position) of TM i n the t h i n f i l a -ment . L i n (39) found that the s u l f h y d r y l s p e c i f i c probe N-(1-pyrene)-iodoacetamide (PIA), when bound to TM, d i f f e r e d from the maleimide species. The distance between the s i t e of attachment and the pyrene group i n PIA-labelled TM i s reduced by one C-C bond and r o t a t i o n around the C-S bond at the point of attachment i s less s t e r i c a l l y hindered. The r e s u l t s showed that excimer i n PIA-TM i s more stable to denaturation by GuHCl ad d i t i o n than excimer i n PM-TM. Burtnick et a l . (40) used PIA to l a b e l p l a t e l e t TM (P-TM) to study i t s r e l a t i v e a b i l i t y to i n t e r a c t with other TM species and i t s a c t i n binding properties. M i l d r e a c t i o n conditions that would only allow a l i m i t e d degree of re a c t i o n of PIA at Cys-190 of muscle TM (0.1 pyrenes/TM chain) gave an average of 1.12 pyrene groups per P-TM chain. The prime s i t e of l a b e l l i n g was not Cys-153 (the homologue of Cys-190), but rather Cys-246, the penultimate COOH-terminal residue. The emission of Py-P-TM ( s o l i d l i n e , Figure 14) shows that the bulk of the pyrenes were i n excimer configuration with the r e l a t i v e heights of the 485 and 385 nm peaks (F485/F385) approaching 4, compared to 1.4 f o r muscle TM l a b e l l e d with PIA (39) and 0.4 f o r muscle TM l a b e l l e d with PM (8). I f the repeating heptapeptide scheme for tropomyosin (Figure 7) i s applied to P-TM, i t i s found that Cys-246 occupies p o s i t i o n 4, hence re q u i r i n g considerable f l e x i b i l i t y of the COOH-terminii of the i n d i v i d u a l chains f o r excimer formation ( t h i s also explains the r e l a t i v e ease of l a b e l -- 29 -i 1 1 1 1 1 r 360 420 480 540 Wavelength (nm) F i g . 14: Emission s p e c t r a of Py-P-TM. S o l i d l i n e , n a t i v e Py-P-TM; dashed l i n e , same Py-P-TM a f t e r treatment w i t h t r y p s i n f o r 6 hours a t 37°C; dotted l i n e , P-TM t r e a t e d w i t h carboxypepti-dase and subsequently l a b e l l e d w i t h PIA (40). - 30 -ling). This f l e x i b i l i t y is restricted when Py-P-TM binds to actin and polymerizes along the F-actin filament as reflected in a drop in rela-tive excimer fluorescence, an effect that levels off at about 6 G-actins per P-TM. This i s consistent with cosedimentation evidence that P-TM could interact with muscle actin at a mole ratio of 1 to 6, as expected from i t s reduced length relative to S-TM (32). Figure 15 shows the effect of various TM species on the ^485/^385 ratio in Py-P-TM in low salt solutions. Non-polymerizable TM (NPTM; C-TM with modified COOH-terminus) produces a greater reduction in 0 1 2 3 4 5 [ T M ] / [ P y - T M ] F i g . 15: Additi o n of un l a b e l l e d TM's to Py-P-TM and e f f e c t on F 4 8 5 / F 3 8 5 . (•) P-TM; (X) C-TM; (+), NPTM. 0 and • and • represent values measured a f t e r a d d i t i o n of KC1 to 275 mM to the P-TM, C-TM and NPTM samples, r e s p e c t i v e l y (40). - 31 -F485/ F385 t t i a n C " ™ because at a given [TM] , more NR^-termini of NPTM would be a v a i l a b l e to complex with Py-P-TM, as s e l f polymerization i s not p o s s i b l e . P-TM shows l i m i t e d i n t e r a c t i o n with Py-P-TM as r e f l e c t e d i n a s l i g h t drop i n F 4 8 5 / F 3 8 5 r a t i o . This, and the other decreases i n F485/ F385 induced by C-TM and NPTM could be reversed by a d d i t i o n of s a l t to disrupt the end-to-end i n t e r a c t i o n s . The addition of s a l t to Py-P-TM alone also increased the F 4 8 5 / F 3 8 5 r a t i ° but not as much as Graceffa and Lehrer (29) observed for muscle TM. This may be because a larger p o r t i o n of the t o t a l emission from Py-P-TM i s excimer i n i t i a l l y . Hence, from these r e s u l t s i t can be concluded that the COOH-terminus of P-TM can more r e a d i l y i n t e r a c t with the N H 2 -terminus of C-TM than with the N H 2 -terminus of P-TM. This may be a r e s u l t of the a d d i t i o n a l residues at the N^-terminus of P-TM that are not present i n muscle TM ( 3 4 ) . In addition, the COOH-terminus of P-TM i s strongly homologous with the COOH-terminus of smooth muscle TM (41, 42), which r e a d i l y undergoes end-to-end i n t e r a c t i o n i n low i o n i c strength solu-tion s , and therefore i t can be postulated that the COOH-terminus of P-TM may i n t e r a c t better with the N H 2 -terminus of a muscle TM than would the N H 2-terminus of P-TM with the COOH-terminus of any other TM. - 32 -B. MATERIALS AND METHODS PART 1: PROTEINS 1.1 Platelet Preparation We used the method of CfSte' et a l . (31, 43) to prepare p l a t e l e t s from f r e s h horse blood. Blood (120 l i t r e s ) was c o l l e c t e d at Alsask Processors, Edmonton, d i r e c t l y from f r e s h l y k i l l e d horses into three large, p l a s t i c p a i l s , each containing 9 l i t r e s of anticoagulant (720 g, NaCitrate, 89 g c i t r i c acid, 61 g Na^PO^, 1400 g dextrose dissolved to 27 l i t r e s with water). This was s t i r r e d to completely d i s t r i b u t e the anticoagulant through the blood, thus preventing rupture of the plate-l e t s . The blood was then transported d i r e c t l y to the laboratory and allowed to s e t t l e f o r 1 hour. Due to the density of the red c e l l s i n horse blood r e l a t i v e to the plasma, two layers quickly form i n the p a i l s , with a p l a t e l e t - r i c h plasma layer on top which was subsequently siphoned o f f . To remove most of the remaining red c e l l s , the plasma was c e n t r i -fuged at a r e l a t i v e l y slow speed (1000 rpm) f o r ten minutes. The supernatant was then centrifuged at 5000 rpm f o r ten minutes to p e l l e t out the crude p l a t e l e t s . The f i n a l p u r i f i c a t i o n of the p l a t e l e t s involved a d i f f e r e n t i a l l y s i s of unwanted c e l l s . The p l a t e l e t p e l l e t s were suspended i n a washing buffer (0.9% w/v NaCl, 0.3% Na c i t r a t e , 1 mM EDTA, 2.5 mM DTT i n 1 l i t r e of water) to which was then added water 33 -(1.5 x volume of buffer used). This was s t i r r e d f o r 2 minutes during which time any remaining red c e l l s were lysed due to there being a large osmotic pressure gradient across the c e l l membranes — p l a t e l e t s can stand t h i s gradient for short periods and hence are not lysed. The l y s i s was terminated by addition of 3.5% NaCl (40% of o r i g i n a l b u f f e r volume) and the f i n a l s o l u t i o n was centrifuged at 8000 rpm for 10 minutes. A two layer p e l l e t was produced, with clean p l a t e l e t s on top and a small amount of red blood c e l l s and l y s i s debris below. The clean p l a t e l e t s were removed and freeze dried. The y i e l d per 100 l i t r e s of blood was approximately 30 g of p l a t e l e t s . 1.2 P u r i f i c a t i o n of Proteins P l a t e l e t TM was prepared according to the method of Cote et a l . (31, 43) In the p u r i f i c a t i o n of P-TM, i t i s necessary to f i r s t homogen-ize the p l a t e l e t s and extract them with pH 2 s o l u t i o n (Figure 16). Under these conditions, the c o i l e d c o i l of TM i s stable but many other proteins present are denatured. When the pH of the pooled extracts i s adjusted to 4.4, the extracted TM p r e c i p i t a t e s out. This provides a greater than lOx p u r i f i c a t i o n of the P-TM i n a s i n g l e step. A f t e r resuspension i n a pH 7.8 buffer and a second pH 4.4 p r e c i p i t a t i o n , the P-TM i s almost 60% pure. The next step, which involves heating the P-TM s o l u t i o n i n a b o i l i n g water bath to 70°C and subsequent recooling, removes most of the remaining impurities. A f i n a l p u r i f i c a t i o n on a hydroxylapatite column, - 34 -11 g P l a t e l e t s ( l y o p h i l i z e d ) C e n t r i f u g e > F i r s t e x t r a c t (10,000 x g) f o r 10 mins I P e l l e t E x t r a c t w i t h 1 l i t r e o f 0.1 M NaCl, 2.5 mM EDTA, 25 mM HC1, pH 2, f o r 1 hour a t 4°C + I n h i b i t o r s (100 til o f 2 mg/ml l e u p e p t i n i n H2O, 100 ul o f 2 mg/ml p e p s t a t i n i n DMSO, 35 mg PMSF i n 1 ml MeOH) I E x t r a c t w i t h 500 ml o f 50 mM HC1 j f o r 30 mins. a t 4°C C e n t r i f u g e > Second e x t r a c t (10,000 x g) f o r 10 mins P e l l e t s ( d i s c a r d ) I P o o l F i r s t and Second E x t r a c t s I A d j u s t pH t o 4.4 ( w i t h 1.0 M NaOH) si/ C e n t r i f u g e > D i s c a r d s u p e r n a t a n t (5,000 x g) f o r 10 mins. F i r s t pH 4.4 p r e c i p i t a t e Fig. 16: continued - 35 -| Dissolve i n 600 ml of 0.1 M NaCl, | 10 mM T r i s , 2 mM DTT, 2.5 mM EDTA, j pH 7.8. S t i r f o r 1 hour at 4°C Centrifuge > Discard p e l l e t (16,000 x g) f o r 10 mins. Supernatant I | Adjust to pH 4.4 (with 1.0 M HCl) Centrifuge > Discard supernatant (5,000 x g) for 10 mins. Second pH 4.4 p r e c i p i t a t e | Dissolve i n 25 ml of 1.0 M KC1, 1 j mM KH 2P0 4/K 2HP0 4, 0.25 mM DTT, pH 7 ^ Heat to 70°C i n b o i l i n g H 20 bath. Centrifuge > Discard p e l l e t (16,000 x g) for 10 mins. \1/ Supernatant I Chromatography on Hydroxylapatite I -30 mg P-TM Fig 16: Purification of Platelet TM 36 -using a phosphate gradient to elute the proteins, y i e l d s P-TM greater than 95% pure as judged by SDS-PAGE. Cardiac and s k e l e t a l TM's were p u r i f i e d , using the methods devel-oped i n the lab of L.B. S m i l l i e (44,45), from acetone powders of the respective muscle ti s s u e s . These acetone muscle powders were prepared v i a several ethanol and acetone extractions of homogenized muscle t i s s u e . The C-TM was obtained from rabbit hearts and the S-TM was obtained from rab b i t back and leg muscle, as was the a c t i n used i n t h i s t h e s i s , which was p u r i f i e d by the method of Spudich and Watt (46). 1.3 Carboxypeptidase A-Treatment of P-TM Mak and S m i l l i e (19) prepared a non-polymerizable form of C-TM by a d d i t i o n of carboxypeptidase A i n an enzyme/substrate weight r a t i o of 1:50 r e l e a s i n g 10 amino a c i d residues from the COOH-terminus of the C-TM. The subsequent loss of h e a d - t o - t a i l p o l y m e r i z a b i l i t y at low s a l t and actin-binding c a p a b i l i t y of the treated C-TM confirmed the impor-tance of COOH-terminus i n these functions of TM. The purpose of t r e a t i n g P-TM with carboxypeptidase A was to remove the cysteine residue at p o s i t i o n 246 and produce a P-TM analogous to C-TM with only one cysteine at p o s i t i o n 153 ( c f . Cys 190 i n C-TM). P-TM (4 mg/ml) was dialyzed against 10 mM t r i s - H C l , 0.15 M KC1, 2 mM DTT, 0.01% sodium azide, pH 8.0 and incubated at 37°C with DFP-treated carboxypeptidase A i n a r a t i o of 1:50. At time =0, 1 hour, 3 hours, and 5 hours, 20 uL aliquots were taken from the reaction - 37 mixture and allowed to react with 20 ul of 10 mM dansyl chloride i n acetone f o r 30 minutes. (Dansyl chloride reacts with free amine groups and hence with any free amino acids i n the s o l u t i o n and with l y s i n e and arginine residues on the P-TM chain). These solutions were then sub-j e c t e d to TLC on polyamide layer sheets i n 3 solvent systems (47); 1. Formic a c i d 1.5% (v/v) i n water, 90° r o t a t i o n then 2. Benzene:AcOH (9:1) 3. E t h y l acetate:AcOH:MeOH (20:1:1) Using a hand-held u l t r a v i o l e t l i g h t source, the r e s u l t i n g fluores-cent spots on the TLC plates were compared to those of pure dansyl amino acids (48). I t was apparent that the carboxypeptidase A had released Ile-247 and Cys-246 a f t e r 1 hour and Asn-245 a f t e r 3 hours. A f t e r 5 hours, a small amount of Leu-244 was seen. The r e a c t i o n was stopped at 6 hours by heating to 85°C for 3 minutes to denature the carboxypepti-dase. The s o l u t i o n was then cooled and centrifuged to remove the p r e c i p i t a t e d carboxypeptidase A. 1.4 Fluorescent Labelling of P-TM P-TM was dialyzed overnight i n 10 mM t r i s - H C l , 0.15 M KC1 and 2.0 mM DTT, pH 8.0 and then for 4-5 hours i n the same buffer without DTT. N-(l-pyrene)iodoacetamide (PIA) (Molecular Probes) was dissolved i n N,N-dimethylformamide and added slowly to the s t i r r i n g TM sample u n t i l an approximate lOx molar excess of reagent to p r o t e i n was achieved. The - 38 -rea c t i o n was allowed to proceed overnight at room temperature i n the dark on a mechanical rocker. The reaction mixture was centrifuged at 15,000 x g for 10 minutes to remove the p r e c i p i t a t e d reagent and the supernatant d i a l y z e d against 10 mM t r i s - H C l , 0.15 M KCl, pH 8.0, to remove the l a s t traces of excess reagent. Reaction conditions were e s s e n t i a l l y the same f o r acrylodan (Molec-u l a r Probes) except that a suitable degree of l a b e l l i n g could be attained a f t e r 1-4 hours at 4°C. 1.5 Extent of Labelling The extent of l a b e l l i n g of P-TM with fluorescent reagents was quantitated by independent determination of the l a b e l and pr o t e i n i n the sample. Acrylodan and pyrene concentrations were determined spectropho-t o m e t r i c a l l y using molar absorption c o e f f i c i e n t s of 1.29 x lO^M^cm'^ (2) and 2.2 x loSl'^-cm"! (51), r e s p e c t i v e l y . The concentration of the l a b e l l e d p r o t e i n was measured using the Bio-Rad assay based on a dye binding assay developed by Bradford (49) . Absorbance was measured at 595 nm where the bound dye absorbs. A standard graph of absorbance vs. [unlabelled S-TM] i n the presence of dye allowed the concentration of l a b e l l e d P-TM to be calcu l a t e d . Therefore, knowing both the l a b e l and pr o t e i n concentration, i t was possible to determine the average number of fluorescent molecules per P-TM chain. - 39 -1.6 Specificity of Labelling To ensure that the fluorescent reagents were only reacting at the desired s i t e (Cys-246 on P-TM; Cys-153 on carboxypeptidase A-treated P-TM; Cys-190 on C-TM), a t r y p t i c digest of the sample was c a r r i e d out followed by peptide separation on c e l l u l o s e chromatogram sheets v i a electrophoresis i n one dimension and TLC i n a second dimension (50). The sample to be digested was dia l y z e d against 50 mM ammonium bicarbonate f o r two days a f t e r which TPCK-Trypsin was added (1/50 of P-TM weight). This was incubated at 37°C f o r 6 hours. Then the sample was freeze-dried. The product was dissolved i n 100 /xl of water and freeze d r i e d again to remove any l a s t traces of ammonium bicarbonate. The digested sample was then dissolved i n a minimal volume of electrophoresis buffer (5-20 LI! of 10% v/v pyridine i n 0.06 M acetic acid) and spotted about 1/3 of the way along from the p o s i t i v e electrode on a 20 cm x 20 cm c e l l u l o s e TLC sheet. This was electrophoresed f o r 1 1/4 hours at 400 V, a f t e r which the plate was removed and dr i e d over-night. I t was then chromatographed perpendicular to the electrophoresis d i r e c t i o n f o r 2 1/2 hours i n pyridine (5): n-butanol (4):water (4): ac e t i c a c i d (1). A f t e r subsequent overnight a i r drying, the plate could be inspected f o r fluorescent peptides using a hand-held u l t r a v i o l e t lamp. The presence of one fluorescent spot was good evidence that the l a b e l had reached s p e c i f i c a l l y at the cysteine of i n t e r e s t . Occasion-a l l y , a weakly fluorescent spot appeared i n samples of P-TM, i n d i c a t i n g a small amount of l a b e l l i n g at Cys-153. Confirmation of the prime s i t e of l a b e l l i n g of P-TM was obtained by scraping o f f the fluorescent spot - 40 -and e x t r a c t i o n of the peptide from the c e l l u l o s e . The extracted peptide was hydrolyzed i n 6N HCl and the hydrolyzate was sent to the lab of Dr. L.B. S m i l l i e at the Un i v e r s i t y of Alberta for amino a c i d a n a l ysis. The r e s u l t s were compared against the known amino a c i d contents of p o t e n t i a l COOH-terminal peptides of P-TM (40). PART 2: OPTICAL METHODS Fluorescence spectra were recorded using a fluorimeter constructed i n our laboratory e s s e n t i a l l y as described by Gafni and Steinberg (52) (Figure 17). E x c i t a t i o n was performed using a 200 W Mercury-Xenon arc lamp (Conrad-Hanovia) through an Applied Photophysics Ltd., f/3.4 grating monochromator with a Schott UG11 black glass f i l t e r at the e x i t port of the monochromator. The e x c i t a t i o n wavelength used i n these studies was e i t h e r 313 nm or 365 nm, indicated i n the appropriate figure legends, with 5 nm band widths. Emission was detected using an EMI 9558 QB photomultiplier tube at 90° or 180° to the e x c i t a t i o n beam, a f t e r passage of the emitted l i g h t through a 1 cm path of a 2 M NaN02 s o l u t i o n , which cuts o f f l i g h t having a wavelength below 400 nm, and a Spex Doublemate double monochromator equipped with a wavelength drive mechanism. (Emission band widths were 10 nm). To increase the si g n a l -to-noise r a t i o , the e x c i t a t i o n beam was modulated at a nominal frequency of 400 Hz using a Photochemical Research Associates (PRA) 301 M modula-tor connected to a PRA M304 power supply. The res u l t a n t 400 Hz fluo r e s -"DC" Vo tmeters Lock-in Amp. (Polariser) PMT Monochromator "AC" J 1 Hre-amp, 4 X-drive Ref, Signal Computer n F i l t e r Lens KH-tf (PEM) Sample A (Pole ^ • ^ L e n s F i l t e r Lens tono-chrom. Lamp 1 (PEM Power Supply) (Polariser) _ F i l t e r Monochromator Lamp 2 Modulator (400Hz) Fig. 17: Set-up for measurement of fluorescence intensities with additional features required for polarization measurements given in brackets. PEM-Photo-Elastic Modulator, PMT-Photo Multiplier Tube. The optical axis of the PMT was vertical and the axes of the excitation and emission polarizers were +45° and -45°, respectively, during polarization experiments. Modulation of lamp 2 was not performed during polarization measurement. - 42 -cence emission s i g n a l was detected and amplified using a Princeton Applied Research model 121 l o c k - i n a m p l i f i e r . The output s i g n a l was d i g i t i z e d and stored on disk using an Apple H e computer. P o l a r i z a t i o n values were obtained by incorporation of u l t r a v i o l e t -p o l a r i z i n g f i l m s (Polacoat) and a photoelastic l i g h t modulator (Hinds International) into the fluorimeter (53) (bracketed terms i n Figure 17). Two spectra were recorded simultaneously from the photomultiplier, one being the DC current that had been converted to a voltage i n the pre-amp and the other being the AC current r e f l e c t i n g the frequency of modula-t i o n which i s also converted to a voltage i n the pre-amp and detected by the l o c k - i n a m p l i f i e r with the a i d of a reference s i g n a l from the modulator power supply. For head-on e x c i t a t i o n , AC a (I||-I±) and DC a AC oAC/V (I I| + I I ) , g i v i n g p - F — and f o r 90° e x c i t a t i o n , p - ^ (53). F DC F - A C / D C i s an experimentally obtained constant and can be c a l c u l a t e d using p -0.4 for f l u o r e s c e i n (10" 5 M) i n 95% g l y c e r o l at 10°C at 500 nm a f t e r e x c i t a t i o n at 365 nm (54). Fluorescence l i f e t i m e s were measured i n the lab of Dr. Ian Soutar at Heriot-Watt U n i v e r s i t y , Edinburgh, on an Edinburgh Instruments 199 s i n g l e photon counting fluorimeter. The decay p r o f i l e i s b u i l t up on a multichannel analyzer and the best exponential decay i s f i t t e d to the p r o f i l e by computer, a f t e r the lamp pulse has been e l e c t r o n i c a l l y deconvoluted to give the true fluorescence response. - 43 -C. RESULTS AND DISCUSSION PART 1: EXCIMER FLUORESCENCE OF PYRENE-LABELLED PLATELET TROPOMYOSIN (PY-P-TM) 1.1 Incorporation of Label The degree of l a b e l l i n g of P-TM was 1.12 ± 0.37 pyrene groups per chain, based on 9 l a b e l l i n g experiments. This was s i g n i f i c a n t l y higher than the maximum value of 0.1 moles of pyrene per chain on C-TM under i d e n t i c a l l a b e l l i n g conditions ( r e c a l l i n g that Cys-190 on C-TM occurs at p o s i t i o n 2 i n the heptapeptide sequence; Figure 7). The reducing conditions of the buffer (2 mM DTT) were such that any cystine groups at p o s i t i o n 246 i n P-TM could be reduced to two cysteines on adjacent chains r e l a t i v e l y e a s i l y as compared to Cys-190 of C-TM or Cys-153 of P-TM. The f a c t that Cys-246 i s the penultimate COOH-terminus residue and hence more accessible to DTT may explain why a s i g n i f i c a n t degree of l a b e l l i n g i s achieved r e l a t i v e to C-TM. 1.2 D i l u t i o n E f f e c t Spectra s i m i l a r to those i n Figure 14 can be used to observe changes i n excimer to monomer peak heights to y i e l d information as to the e f f e c t of v a r i a b l e conditions such as s a l t concentration and added p r o t e i n on the conformation of the COOH-terminus of P-TM. - 44 -I t was found that i f the 10'^mol 1'^, there was an increase was not expected, shown i n Table 1 Py-P-TM concentration was above -4 x i n excimer with concentration that Table 1 Sample Concentration (Mol 1 ) F485/F402 2. ,89 X 10-5 8. ,28 1. .45 X 10-5 7. .69 7, .23 X i c r 6 5, .92 3 .61 X i c r 6 5, .76 Previous experiments on the d i l u t i o n e f f e c t on Py-P-TM samples ( 5 5 ) showed no dependence of F 4 8 5 / F 4 0 2 r a t i ° o n concentration but these were c a r r i e d out using stock solutions that were less than 4 x 1 0 " ^ mol 1"^. At higher concentrations, i t i s possible that there i s some intermolecu-l a r excimer formation which can be eliminated by d i l u t i o n , which would lower the p r o b a b i l i t y of 2 TM molecules "stacking" and forming excimer between pyrenes on adjacent chains. This alone would not explain the increase i f a l l the pyrenes were previously involved i n intramolecular excimer because these would have to be broken before an intermolecular excimer could be formed. However, there i s an equilibrium between 45 -pyrenes i n the monomer and excimer conformation and i t i s possible for monomer pyrenes to i n t e r a c t with pyrenes on other molecules to increase the r a t i o i n favour of excimer. This along with intermolecular excimer formation between pyrenes on s i n g l y - l a b e l l e d P-TM molecules, which are not capable of intramolecular excimer formation, can explain the increase i n excimer above [Py-P-TM] of -4 x 10"^ mol 1"^ and led to a l l experiments i n t h i s thesis being done at [TM] = 2 x 10"^ mol 1"^ or lower. P-TM 1.3 Varying Keeping [TM] T n 1- Constant Py-P-TM l o z Working at a constant [TM] = 2 x 10"^ mol 1"^, samples with varying r a t i o s of P-TM to Py-P-TM were prepared. Because the t o t a l concentra-t i o n of l a b e l l e d and unlabelled TM was held constant, there should have been no change i n the r a t i o of excimer to monomer emission. However, the extent of excimer fluorescence was found to be l e a s t i n solutions that contained the highest mole r a t i o of unlabelled to l a b e l l e d P-TM (Figure 18). This suggests that the two forms of P-TM are not i d e n t i c a l i n t h e i r tendencies to polymerize and that the pyrene groups disrupt the end-to-end i n t e r a c t i o n . I s h i i and Lehrer (38) reported s i m i l a r disrup-t i v e e f f e c t s of pyrene on muscle TM at Cys-190 and showed that F-actin binding was i n h i b i t e d , p r i m a r i l y by reduced end-to-end TM inte r a c t i o n s on the a c t i n filament. I t i s i n t e r e s t i n g to note that d i s r u p t i o n of the c o i l e d c o i l i n the c e n t r a l region of the muscle TM can have a profound e f f e c t on the end(s) of the molecule to the extent that i t s a b i l i t y to - 46 -CVJ o Ta-l l -i n oo 0.1 1.0 1 0 . 0 [P-TM] / [Py-P-TM] F i g . 18: I n t e r a c t i o n of u n l a b e l l e d P-TM w i t h Py-P-TM. Samples were d i a l y z e d a g a i n s t 10 mM T r i s - H C l , pH 8.0. Spectra were recorded before (X) and a f t e r (+) the a d d i t i o n o f KC1 t o 390 mM. E x c i t a t i o n was at 313 nm. polymerize i s reduced (22,29,39). There i s however, an important d i f f e r e n c e i n the e f f e c t the l a b e l has on the p l a t e l e t and muscle TM's. I n the case o f muscle TM, the presence o f pyrenes a t Cys-190 causes a 10% l o s s o f h e l i c i t y (as meas-- 47 -ured by c i r c u l a r dichroism) whereas pyrenes on Cys-246 of P-TM cause no apparent loss of h e l i x and are probably a f f e c t i n g the end-to-end i n t e r -a c t i o n i n a s t e r i c manner by t h e i r p h y s i c a l presence at the COOH-terminus of P-TM. I t can be seen from Figure 18 that the addition of KC1 to 390 mM causes depolymerization, allowing the reformation of intramolecular excimers i n Py-P-TM. Maximal excimer to monomer r a t i o i s not achieved for the solutions with high unlabelled to l a b e l l e d P-TM, however, suggesting that there i s some end-to-end i n t e r a c t i o n remaining. C-TM 1.4 Varying Keeping [TM] T o 1_ Constant Py-P-TM l o t The previous experiment was repeated using C-TM instead of P-TM i n order to ensure that the decrease i n F435/F335 upon C-TM addition observed by Burtnick et a l . (40) was not dependent on t o t a l p r o t e i n concentration. The r e s u l t s , (shown i n Figure 19) i n d i c a t e that as the C-TM to Py-P-TM r a t i o i s increased, the excimer con t r i b u t i o n drops o f f with a concommitant increase i n monomer emission. This confirms that there i s a more favourable i n t e r a c t i o n between the COOH-terminus of P-TM molecule and the NH2-terminus of C-TM than between the COOH-terminus of P-TM and the NH2-terminus of P-TM. This may be due to a d d i t i o n a l residues at the NH2-terminus of P-TM that are not present i n muscle TM (34). Also, the COOH-terminus sequence of P-TM i s strongly homologous to the COOH-terminus of smooth muscle TM (41,42) which r e a d i l y undergoes end-to-end polymerization i n low i o n i c strength s o l u t i o n s . This again - 48 -0.1 1.0 10.0 [ C - T M ] / [ P y - P - T M ] F i g . 19: Inte r a c t i o n of unlabelled C-TM with Py-P-TM. Samples were dialyzed against 10 mM Tri s - H C l , pH 8.0. Spectra were recorded before (X) and a f t e r (+) the ad d i t i o n of KC1 to 390 mM. E x c i t a t i o n was at 313 nm. points to the a l t e r e d N H 2 -terminus of P-TM (with respect to muscle TM) as the cause f o r reduced p o l y m e r i z a b i l i t y . As with Figure 18, Figure 19 also shows an increase i n F485/ F402 w n e n s a l t i s added, showing that the end-to-end i o n i c i n t e r a c t i o n s between C-TM and Py-P-TM are disrupted. Again, at the highest C-TM/Py-P-TM r a t i o , the added KC1 does not break - 49 -a l l the end-to-end i n t e r a c t i o n s . (To check that there was no d i l u t i o n e f f e c t when the KC1 was added (150 fil to a 1 ml sample), the 3/1 s o l u t i o n was selected and made up to 1 ml with 390 mM KC1 i n the so l u t i o n . The F485/F402 r a t i o f o r t h i s s o l u t i o n was the same as for the s o l u t i o n which was o r i g i n a l l y 1 ml and had KC1 added.) At t h i s stage, the recently developed fluorescent probe, acrylodan (2), showed that i t had several properties making i t very a t t r a c t i v e f o r the study of P-TM: a) I t i s s u l f h y d r y l - s p e c i f i c , allowing l a b e l l i n g at Cys-246. b) I t has a l i f e t i m e of 1.65 ns, making p o l a r i z a t i o n studies po s s i b l e . c) I t i s s e n s i t i v e to the p o l a r i t y of i t s environment. PART 2: FLUORESCENCE OF ACRYLODAN-LABELLED PLATELET TROPOMYOSIN (AD-P-TM) 2.1 Incorporation of Label L a b e l l i n g of P-TM with acrylodan could be v a r i e d from 0.5 to 1.3 AD molecules per P-TM chain by varying reaction time from 1 to 5 hours at 4°C. When C-TM or carboxypeptidase A-treated P-TM were reacted with AD, s i g n i f i c a n t degrees of l a b e l l i n g required stronger reducing conditions - 50 -(up to 10 mM DTT) (56) due to the r e l a t i v e i n a c c e s s i b i l i t y of the S-S bonds on these proteins with respect to Cys-246 on P-TM. Degrees of l a b e l l i n g i n these cases reached 0.4 AD per C-TM chain and 0.7 AD per truncated P-TM chain. Comparison of the c i r c u l a r dichroism spectra of AD-P-TM and unla-b e l l e d P-TM over the region from 200 to 250 nm showed no s i g n i f i c a n t d ifferences i n d i c a t i n g that the incorporation of the l a b e l d i d not produce a detectable unfolding of the TM molecule. 2.2 Emission Properties On e x c i t a t i o n at 365 nm, AD-P-TM i s high l y fluorescent, showing a broad emission band with a maximum near 520 nm (Figure 20). Similar spectra are obtained with AD-C-TM and AD-labelled truncated P-TM. The emission maximum i s b l u e - s h i f t e d r e l a t i v e to an AD-mercaptoethanol adduct (540 nm), but s i g n i f i c a n t l y to the red of AD-labelled proteins papain . (491 nm), parvalbumin (498 nm) and carbonic anhydrase (501 nm) (2). The p o s i t i o n of the emission maximum of AD-P-TM demonstrates that, while AD i s i n a more hydrophobic environment r e l a t i v e to when bound to mercaptoethanol, the probe i s r e l a t i v e l y exposed to the solvent when compared to the binding s i t e s i n the aforementioned proteins which are known to have considerable hydrophobic character. This i s not unex-pected considering that P-TM i s an extended c o i l e d c o i l and w i l l not a f f o r d a great deal of protection from the solvent, no matter what part of the molecule the probe i s attached to. - 51 -4 6 0 Fig. 20: 5 0 0 W A V E L E N G T H ( n m ) 5 4 0 Emission spectrum of AD-P-TM i n 300 mM KC1, 20 mM MOPS, pH 7.0 (dashed l i n e ) ; S o l i d l i n e i s buffer alone. E x c i t a t i o n was at 365 nm. 2.3 Denaturation of AD-P-TM with Guanidine Hydrochloride (GuHCl) This preliminary experiment was c a r r i e d out to ensure that the AD was s e n s i t i v e to changes i n the COOH-terminus of P-TM In terms of r i g i d i t y and p o l a r i t y of the probe s i t e . Two solutions of AD-P-TM were prepared, one i n low s a l t (20 mM MOPS) and one i n high s a l t (0.3 M KC1, - 52 -20 mM MOPS). To these were added aliquots of guanidine hydrochloride (8 M) to a f i n a l concentration of 4 M. A f t e r each ad d i t i o n the fluores-cence p o l a r i z a t i o n and i n t e n s i t y were measured at 520 nm (Figure 21). The r e s u l t s show that as guanidine hydrochloride disrupts the hydropho-b i c i n t e r a c t i o n s that are holding the c o i l e d c o i l together, so the i n t e n s i t y and p o l a r i z a t i o n of the acrylodan emission decrease r e f l e c t i n g that the COOH-terminus has become uncoiled. The r i g i d i t y of the i n d i -v i d u a l strands of P-TM i s much reduced r e l a t i v e to the i n t a c t p r o t e i n as expected and t h i s i s borne out by the p o l a r i z a t i o n drop. Also, as the c o i l i s disrupted at the COOH-terminus, the acrylodan becomes more exposed to solvent c o l l i s i o n s which r e s u l t i n a net decrease i n f l u o r e s -cence i n t e n s i t y as r a d i a t i o n l e s s deactivation increases. At the f i n a l concentration of guanidine hydrochloride of 4 M, there i s a higher r e s i d u a l p o l a r i z a t i o n i n the high s a l t sample because the KC1 s t a b i l i z e s the c o i l e d c o i l v i a strengthened hydrophobic i n t e r a c t i o n s and hence a higher concentration of denaturant would be required to reach the same stage of denaturation as i n the low s a l t sample. The i n t e n s i t y p l o t s are the same i n low and high s a l t suggesting that the COOH-terminus has completely uncoiled at 4 M GuHCl. 2.4 Thermal Denaturation of AD-TM Species - P o l a r i z a t i o n Studies P e r r i n p l o t s of p o l a r i z a t i o n data for d i f f e r e n t AD-labelled TM species are shown i n Figure 22. AD-P-TM was l a b e l l e d p r i m a r i l y at Cys-246. Therefore, a f t e r d i a l y s i s against buffers devoid of d i s u l f i d e -- 53 -e 266 400 600 Added GuHCl(pi) 800 Added GuHCl(ul) 200 400 666 Added GuHCl(ul) 860 d) .34 . 3 0 -.26-. 2 2 -. 1 8 -266 400 660 Added GuHCl(ul) 860 F i g . 21: Change In r e l a t i v e fluorescence i n t e n s i t y a t 520 nm ( F 5 2 0 ) A N D p o l a r i z a t i o n (p) w i t h a d d i t i o n of GuHCl t o samples i n 20 mM MOPS, pH 7.0 (a and c) and 300 mM KC1, 20 mM MOPS, pH 7.0 (b and d). E x c i t a t i o n was at 365 nm. 54 T/T) (K/poise)xlO -4 F i g . 22: Pe r r i n p l o t s of AD-P-TM i n 20 mM MOPS, pH 7.0, i n the absence ( s o l i d c i r c l e s ) and presence (open c i r c l e s ) of 300 mM KC1. Pe r r i n p l o t s of AD-C-TM ( s o l i d diamonds) and AD-labelled truncated P-TM (open squares) i n 20 mM MOPS, pH 7.0. The temperature range covered by the AD-P-TM p l o t i s 10-74 CC. Emission was monitored at 517 nm and e x c i t a t i o n was at 365 nm. reducing agents, the predominant form of AD-P-TM present would be cross-l i n k e d at Cys-153 and not cr o s s - l i n k e d at Cys-246. P e r r i n p l o t s for AD-P-TM i n low (20 mM MOPS, pH 7.0) and high (0.3 M KC1, 20 mM MOPS, pH 7.0) i o n i c strength solutions are in d i s t i n g u i s h a b l e . They c l o s e l y - 55 -resemble, i n shape, P e r r i n p l o t s presented by Lehrer (23) f o r the temperature-dependence of p o l a r i z a t i o n of i n t r i n s i c tyrosine fluores-cence from cr o s s - l i n k e d s k e l e t a l muscle TM i n a high i o n i c strength b u f f e r . This agreement i n shape, considering the d i f f e r e n t sources of the fluorescence ( s i x Tyr residues along the TM vs. AD at the p e n u l t i -mate residue), suggests that the p r e t r a n s i t i o n near 30°C causes subtle changes i n structure along tropomyosin that can be detected as f a r away as the ends of the molecule. Evidence f o r transmission of s t r u c t u r a l perturbations down the TM chains also comes from observations that muscle TM modified at Cys-190 with fluorescent l a b e l s a f f e c t the a b i l -i t i e s of TM to polymerize i n low s a l t buffers and to bind to F-actin (29,38,57). The change i n slope of the plot s for AD-P-TM near 50°C also c o r r e l a t e s with the main chain opening t r a n s i t i o n of unlabelled P-TM observed by c i r c u l a r dichroism measurements (31) . The increased slope i n the P e r r i n p l o t above t h i s temperature r e f l e c t s the onset of an ad d i t i o n a l d epolarizing mode, the u n c o i l i n g of the c o i l e d c o i l at the COOH-terminus. Below -50°C, the reason f o r the slow increase of ^/p with i s due to the thermal motion of the solvent inducing increased "wagging" of the i n t a c t COOH-terminus and r o t a t i o n about the long axis of the molecule which reduce AD p o l a r i z a t i o n . End-over-end tumbling i s not a cause of de p o l a r i z a t i o n as i t i s on the microsecond timescale (58). P e r r i n p l o t s f o r AD-C-TM and AD-labelled truncated P-TM resemble each other more c l o s e l y than they resemble that f o r AD-P-TM (Figure 22). This i s due to the absence of Cys-246 i n the truncated P-TM and the incorporation of the l a b e l at Cys-153, the homologue of Cys-190 i n C-TM. - 56 -(Note that the fluorescence s i g n a l w i l l a r i s e from that p o r t i o n of the TM population that i s l a b e l l e d and, therefore i s not cro s s - l i n k e d ) . These p l o t s resemble c l o s e l y those presented by Lehrer (23) f o r P e r r i n p l o t s of i n t r i n s i c tyrosine fluorescence from muscle TM's reduced and iodoacetamide-blocked at Cys-190. The lower thermal s t a b i l i t y of the c o i l e d c o i l i n t h i s region of the molecule i s manifested i n the lower p o l a r i z a t ion values for AD-C-TM and AD-labelled truncated P-TM r e l a t i v e to AD-P-TM i n the 30-60°C range. For both samples i n t h i s temperature range, the p o l a r i z a t i o n decreases uniformly as the temperature i s increased, the slopes of the P e r r i n p l o t s being s i m i l a r to the slope of AD-P-TM above 50°C. This could be explained by considering the Cys-190 and Cys-153 regions of AD-C-TM and AD-labelled truncated P-TM respec-t i v e l y as having separated at -30°C and becoming larger sections of random c o i l which are able to depolarize the fluorescence almost as e f f i c i e n t l y as when the probe i s attached to a sing l e stand of P-TM at Cys-246 above 50°C. The source of the observed p r e t r a n s i t i o n and reduced thermal s t a b i l i t y i n cr o s s - l i n k e d TM's and i n non-cross-linked TM's i n high i o n i c strength buffers (21) i s not f u l l y resolved. There i s agreement that the region near Cys-190 i n muscle TM's i s conformationally s t r a i n e d (23,36,60). This i n s t a b i l i t y near Cys-190 may be the f i r s t to u n c o i l i n response to high temperature or the presence of denaturants giving r i s e to a loop or "bubble" of random c o i l i n the c o i l e d c o i l that expands outwardly to the ends of the molecule (38). A l t e r n a t i v e l y , Skolnick and Holtzer (28) have proposed that the s t r a i n i n C-TM cross-linked at Cys-190 causes u n c o i l i n g of the c o i l e d c o i l inwardly from the COOH-- 57 -terminus. Their theory predicts that the large closed loop formed by a bubble expanding outwardly from Cys-190 would not be s u f f i c i e n t l y stable to grow out to the ends of the molecule. The p o l a r i z a t i o n data pre-sented here f o r AD-P-TM does not seem to f i t t h i s model. I f P-TM uncoiled from the COOH-terminus, the p o l a r i z a t i o n from a probe at Cys-246 should have dropped to near random c o i l values at temperatures j u s t above 30°C with, perhaps, a further s l i g h t drop at the main chain unfolding temperature. Yet, the AD data p a r a l l e l s the c i r c u l a r dichroism and tyrosine fluorescence that a r i s e from signals a l l along the length of the TM molecule. A preliminary unfolding event i s observed at a region away from the l a b e l on Cys-246, followed by increased u n c o i l i n g . The observation of a major loss i n p o l a r i z a t i o n beginning near 50°C, the temperature corresponding to the main melting t r a n s i t i o n temperature of TM, suggests that the region towards the COOH-terminus i s among the l a s t to lose i t s h e l i c a l character. Rigorous a p p l i c a t i o n of the theory put forward by Skolnick and Holtzer to the exact amino a c i d sequence of P-TM may provide a l t e r n a t i v e explanations, as t h e i r theory was based on the C-TM amino a c i d sequence. 2.5 Thermal Denaturation of AD-TM Species and Acrylodan Lifetimes In the course of t h i s project, l i m i t e d access was obtained to a s i n g l e photon counting, fluorescence l i f e t i m e instrument, which allowed r e p e t i t i o n of the experiments i n Section 2.4 but instead of measuring the p o l a r i z a t i o n of the emission, the fluorescence l i f e t i m e s of the 58 -AD-TM s p e c i e s were measured a t v a r i o u s t e m p e r a t u r e s . The r e s u l t s a r e summarized i n T a b l e 2 and i n F i g u r e s 23 and 24. Table 2 Sample Temperature (°C) (ns) r 2 (ns) \ R e l a t i v e A m p l i-tudes o f and r 2 AD-DTT 25 1.78 25 1.02 50 0.96 AD-C-TM 29.7 2.76 29.7 1.79 39.9 2.74 49.6 2.07 55.3 1.82 60.0 3.2 65.0 3.05 A D - l a b e l l e d 30.4 2.82 T r u n c a t e d 34.5 2.78 P-TM 39.9 2.26 42.9 2.45 45.8 2.53 48.8 2.39 52.2 1.81 55.2 2.15 58.7 3.1 63.0 2.44 AD-P-TM 30.2 3.61 34.8 3.39 40.1 2.67 45.0 2.73 49.7 2.45 53.3 2.72 56.1 2.35 59.5 2.69 64.1 2.9 0.59 1.79 0.32:0.68 12.27 1.00 ( s i n g l e expo-n e n t i a l f i t ) 0.3 1.84 0.49:0.51 0.81 2.46 0.48:0.52 11.50 1.00 ( s i n g l e expo-n e n t i a l f i t ) 0. 73 1. ,55 0. ,40: 0. 50 0. 38 4. .14 0. ,45: 0. 55 0. 42 5. ,45 0. ,38: 0. 62 0. .73 1. .99 0. ,30: 0. 70 0. 63 1. ,72 0. ,30: 0. 70 0. .81 1. .37 0. .47: :0. ,53 0. .67 1, .62 0. .44: :0. ,56 0. ,45 2, .84 0, .46: :0. ,54 0. ,52 1. .75 0, .46: :0. ,54 0. .63 1. .87 0, .44: :0. ,56 0. .71 1. .87 0, .44: :0. ,56 0. .49 4, .97 0, .60: :0. .40 0. ,47 2, .09 0, .47: :0. ,53 0, .77 1. .54 0, .35: :0. .65 0. .56 1 .58 0 .37: :0. .63 1. .09 1 .46 0 .45 :0. .55 0. .99 1. .29 0 .48: :0. .52 0, .7 1, .54 0 .52: :0, .48 0. .69 1, .62 0 .43: :0. .57 0. .6 2 .00 0 .50: :0, .50 0. .65 1. .42 0 .43: :0, .57 0. .52 1 .78 0 .49: :0, .51 0, .74 1. .69 0 .38: :0. .62 0. .79 2 .04 0 .37: :0. .63 3.7 -3.3 -2.9 -2.5 2.1 ~ 30 40 50 60 70 Temperature (°C) 23: E f f e c t of temperature on AD l i f e t i m e i n AD-C-TM (open t r i a n g l e s ) and AD-labelled truncated P-TM (closed t r i a n g l e s ) In 300 mM KC1, 20 mM MOPS, pH 7.0. Emission was monitored at 510 and e x c i t a t i o n was at 360 nm. 3.7 3.3 -2.9 -2.5 -2.1 30 40 50 60 70 Temperature (°C) 24: E f f e c t of temperature on AD l i f e t i m e i n AD-P-TM i n 300 mM KC1, 20 mM MOPS, pH 7.0. Emission was monitored at 510 and e x c i t a t i o n was at 360 nm. - 61 -When the fluorescence decays were c o l l e c t e d , a computer was used to i t e r a t i v e l y f i t the best exponential curve to the fluorescence decay p r o f i l e at each temperature. (A sample decay and f i t i s shown i n Figure 25). The q u a l i t y of f i t of the curve to the decay i s given by < the F i g ; 25: Computer f i t of best exponential decay ( s o l i d l i n e ) to the experimental data f o r AD-P-TM at 34.8°C (dots) c o l l e c t e d on multichannel analyzer of a sing l e photon counting fluorimeter. - 62 -cl o s e r the x 2 value i s to 1.0, the better the f i t . Table 2 shows that the best f i t i n a l l the decays were double exponentials, giving two fluorescence l i f e t i m e s . I f a single exponential was used, i t gave a very poor f i t (eq. AD-DTT; x 2 = 12.27). Normally a double exponential decay means e i t h e r there are two d i f f e r e n t probes present or the same probe i s i n two environments of d i f f e r i n g p o l a r i t y , but neither of these s a t i s f a c t o r i l y explain the f a c t that the short l i f e t i m e components found here are shorter even than that f or AD i n water (r = 1.28 and 1.65 ns) (63) and also shorter than the lamp-pulse i t s e l f (-1.0 ns). I t may be that they are a r t i f a c t s caused by the computer's i n a b i l i t y to accurately deconvolute the lamp pulse from such rapid decays, but the f a c t that the r e l a t i v e amplitudes of the short and long l i f e t i m e components are almost equal shows that the short component s i g n i f i c a n t l y a f f e c t s c a l c u l a t i o n s . M u l t i p l e l i f e t i m e s have previously been observed for probes bound to muscle TM's (21,39) and i n the case of didansylcysteine (21), the phenomenon was explained as being due to the probe e q u i l i b r a t i n g between a polar and hydrophobic environment. There was a dependence of the f r a c t i o n of long l i f e t i m e components on temperature with a maximum observed around the p r e t r a n s i t i o n temperature (25-35°C) i n the high s a l t sample and a steady decrease with temperature i n the low s a l t sample, i n d i c a t i n g that the probe does not detect any p a r t i a l l y unfolded intermediates i n the low s a l t case. In the case of the AD-TM species, the long l i f e t i m e component showed the temperature dependence of a p o l a r i t y s e n s i t i v e probe. A l i f e t i m e of 1.78 ns was obtained for the AD-DTT adduct at 25°C which agrees w e l l with the value obtained for AD i n water of 1.65 ns. Figures - 63 -23 and 24 again show how s i m i l a r AD-C-TM and AD-labelled truncated P-TM are to each other and that they are d i f f e r e n t from AD-P-TM. The l i f e -times f o r these three species at 30°C were 2.76 ns, 2.82 ns, and 3.6 ns resp e c t i v e l y . A possible reason for the longer l i f e t i m e of AD at Cys-246 i s that the COOH-terminus can un c o i l enough to l e t the two AD molecules l i e inside the c o i l e d c o i l i n a more hydrophobic environment. In the cases of AD on Cys-190 and Cys-153, i t may be too much s t r a i n on the h e l i c e s to allow the probes to achieve a hydrophobic environment which would increase the l i f e t i m e to 3.6 ns, although they are c e r t a i n l y being protected by the pro t e i n as the l i f e t i m e s are longer than for the AD-DTT adduct. As temperature i s increased, AD-C-TM and AD-labelled truncated P-TM show s i m i l a r responses of fluorescence l i f e t i m e , decreasing slowly i n i t i a l l y then f a l l i n g to a minimum around 54°C, where the chains w i l l be completely separated. The subsequent r i s e i n l i f e t i m e above t h i s temperature may mean that the i n d i v i d u a l strands of TM, which are now random c o i l s , are f o l d i n g , p a r t i c u l a r l y i n the Cys-190 region of AD-C-TM (and Cys-153 region of AD-labelled truncated P-TM) to minimize exposure of hydrophobic s i t e s to the solvent, although a l i f e t i m e increase above the main melting temperature was not observed by Betteridge and Lehrer (21). In the case of AD-P-TM, the l i f e t i m e drops o f f sharply below 40"C then s t a b i l i z e s above t h i s temperature. The drop i s consistent with a model of P-TM that has a loose COOH-terminus which would unwind s l i g h t l y as temperature i s increased but, as the p o l a r i z a t i o n data suggests, the COOH-terminus remains e s s e n t i a l l y i n t a c t u n t i l the main melting t r a n s i -- 64 -t i o n . Due t o the time c o n s t r a i n t s on these l i f e t i m e e xperiments, s u f f i c i e n t d a t a p o i n t s c o u l d n o t be c o l l e c t e d to r e l a t e more a c c u r a t e l y , t h e temperature dependence o f the l i f e t i m e d a t a t o t h a t o f the p o l a r i z a -t i o n d a t a t o produce a c l e a r e r p i c t u r e o f the d e n a t u r a t i o n ( a - h e l i x to random c o i l ) p r o c e s s i n P-TM. 2.6 I n t e r a c t i o n o f AD-P-TM w i t h A c t i n Two p o r t i o n s o f a s t o c k AD-P-TM s o l u t i o n i n 150 mM KC1, 10 mM T r i s - H C l , pH 8.0, were each d i l u t e d to 1.0 ml ( f i n a l [AD-P-TM] = 2.0 x 1 0 " 6 M) w i t h 2.0 mM T r i s - H C l , 1.0 mM DTT, 0.2 mM ATP, 0.2 mM C a C l 2 , pH 7.6 ( G - a c t i n d i a l y s i s b u f f e r ) . M g C l 2 was t h e n added t o 5 mM to s p o n t a n e o u s l y p o l y m e r i z e the G - a c t i n t h a t was s u b s e q u e n t l y added t o one o f the s o l u t i o n s , w h i l e an e q u a l q u a n t i t y o f b u f f e r was added t o the o t h e r . From F i g u r e 26 i t can be seen t h a t the AD f l u o r e s c e n c e i n t e n s i t y i n c r e a s e s w i t h a c t i n a d d i t i o n , i n d i c a t i n g t h a t the probe i s i n a more h y d r o p h o b i c environment i n the a c t i n - T M complex than on P-TM a l o n e and t h a t a c t i n may b i n d near t o the AD attachment s i t e on P-TM ( a l s o c o r r o -b o r a t e d by a s h i f t i n e m i s s i o n wavelength maximum from 518 nm to 513 nm) . However, f l u o r e s c e n c e quenching experiments p e r f o r m e d w i t h KI i n the p r e s e n c e and absence o f F - a c t i n ( F i g u r e 27) show t h a t v e r y l i t t l e p r o t e c t i o n from q u e n c h i n g i s o f f e r e d t o AD by the p r e s e n c e o f F - a c t i n . AD-DTT quenching was pe r f o r m e d to show how the probe i s p r o t e c t e d on the P-TM m o l e c u l e r e l a t i v e t o f r e e i n s o l u t i o n . AD may be s e n s i t i v e enough - 65 -1.3 - T • T • • T 1.2 - • I.I - T 1.0 — T T 1 . i i . i . i . i .9 0 2 4 6 8 10 [ACTIN]/ [AD-P-TM] 26: E f f e c t o f a c t i n on the emission i n t e n s i t y o f AD-P-TM. Two samples o f AD-P-TM were made up ([AD-P-TM] - 2 jiM) t o 1 ml i n 2.0 mM T r i s - H C l , 1.0 mM DTT, 0.2 mM ATP, 0.2 mM C a C l 2 , pH 7.6 (G - a c t i n d i a l y s i s b u f f e r ) . MgCl2 was added t o 5 mM. To one sample a l i q u o t s of G-actin were added and t o the other a l i -quots of d i a l y s i s b u f f e r . Each p o i n t represents the r a t i o of the f l u o r e s c e n c e i n t e n s i t i e s a t 515 nm. E x c i t a t i o n was at 365 nm. 3.0 — • • 2.5 — \ 2.0 1 .5 1.0 • • _ • 1 • • • i V 5 ° 0 .2 .4 .6 .8 [KI] CM) F i g . 27: Fluorescence quenching by KI. I d e n t i c a l p a i r s of s o l u t i o n s of AD-P-TM ( t r i a n g l e s ) , a 10:1 mole r a t i o of a c t i n to AD-P-TM (open c i r c l e s ) and an AD adduct of DTT (closed c i r c l e s ) were made up i n 100 mM KC1, 20 mM MOPS, 1.0 mM MgCl2, pH 7.0 so that [AD] = 250 nM i n each case. F values were measured from the s o l u t i o n s to which KI was added and F D values were measured from the s o l u t i o n s to which b u f f e r was added. Measurements done at 517 nm. E x c i t a t i o n was at 365 nm. - 67 -to detect the presence of a c t i n and r e f l e c t t h i s by increased emission i n t e n s i t y and a blue s h i f t i n the emission maximum but i t i s , i n fac t , s t i l l r e l a t i v e l y exposed to quenching by I", even when a c t i n i s bound. KI at 0.6 M depolymerizes F-actin but at lower KI concentrations t h i s e f f e c t cannot be the cause of the s i m i l a r quenching rates (gradients) of AD-P-TM and AD-P-TM + a c t i n . Fluorescence p o l a r i z a i o n values for AD-P-TM increase from 0.34 to 0.43 i n the presence of F-actin (Figure 28), c l e a r l y i n d i c a t i n g binding between the two proteins. The main reason f o r the enhanced p o l a r i z a t i o n i s l i k e l y to be the reduced rot a t i o n a l freedom of the AD-P-TM molecule, but there may be binding of the COOH-terminus to the F-a c t i n filament as well. The sigmoidal shape of the i n t e n s i t y and p o l a r i z a t i o n plots r e f l e c t the cooperative binding of P-TM to a c t i n , although the coopera-t i v i t y i s not as great as for s k e l e t a l TM which binds 600-1200 times more strongly to s i t e s on a c t i n where they are able to i n t e r a c t with an already bound TM, than to an i s o l a t e d s i t e on a c t i n (18,32). The e f f e c t l e v e l s o f f at an [actin]/[AD-P-TM] r a t i o of about 6/1, which i s consis-tent with r e s u l t s from cosedimentation studies reported by Cote and S m i l l i e (32) and with r e s u l t s from excimer fluorescence of Py-P-TM by Burtnick et a l . (40). 2.7 I n t e r a c t i o n of AD-P-TM with C-TM Previous r e s u l t s i n sections 1.3 and 1.4 and from Burtnick et a l . (40) suggested that the COOH-terminus of P-TM was able to i n t e r a c t with - 68 -[ A C T I N ] / [ A D - P - T M j F i g . 28: E f f e c t of a c t i n on AD p o l a r i z a t i o n from AD-P-TM. G-actin and G-actin d i a l y s i s b u f f e r were added i n d i f f e r i n g amounts to 11 samples of AD-P-TM to a f i n a l volume of 1 ml and f i n a l [AD-P-TM] «= 2 uK. MgCl2 was added to 5 mM to polymerize to a c t i n . Emission was monitored at 517 nm. E x c i t a t i o n was at 365 nm. the NH2-terminus of muscle TM i n low s a l t , although the pyrenes at Cys-246 seemed to reduce the a b i l i t y of P-TM to self-polymerize (Figure 18). - 69 -In t h i s experiment, AD-P-TM and C-TM were dialyzed against 10 mM Tri s - H C l , pH 8.0 and mixtures of the two were made up with d i f f e r e n t mole r a t i o s but a constant t o t a l [TM] - 2 /iM. As more C-TM was introduced, the p o l a r i z a t i o n of the AD on Cys-246 of P-TM increased (Figure 29), i n d i c a t i n g that end-to-end i n t e r a c t i o n can occur between -I 0 I LOG([C-TM]/[AD-P-TM] ) F i g . 29: I n t e r a c t i o n of C-TM with AD-P-TM In 20 mM MOPS, pH 7.0. Fluorescence p o l a r i z a t i o n values were recorded at 517 nm. E x c i t a t i o n was at 365 nm. 70 C-TM and AD-P-TM. This introduces constraints on the f l e x i b i l i t y of the COOH-terminus of AD-P-TM. The magnitude of the increase i s much less than that observed when AD-P-TM was bound to F-a c t i n (Figure 28) as the r e s t r i c t i o n of the mobility of the COOH-terminus i s much greater i n the F - a c t i n complex. There was no change i n p o l a r i z a t i o n of AD at a l l r a t i o s of P-TM/ AD-P-TM i n low and high s a l t , which suggests that e i t h e r the AD had blocked any self-polymerization or that i t allows polymerization but does not detect depolymerization when KC1 i s added. I f the l a t t e r i s the case, i t may be that the P-TM polymerizes i n such a way as to leave the l a s t three or four residues of the COOH-terminus free to f l e x and depolarize the AD fluorescence to the same extent as a sing l e AD-P-TM molecule i n s o l u t i o n . This i s something that w i l l be investigated further i n the future using s u l f h y d r y l - s p e c i f i c , photoreactive cross-l i n k i n g agents that w i l l attach at Cys-246 and be i r r a d i a t e d with u.v. l i g h t to induce c r o s s - l i n k i n g . Addition of KC1 to the C-TM/AD-P-TM samples i n Figure 29 caused a drop i n p o l a r i z a t i o n i n each case, consistent with a lower tendency of TM molecules to i n t e r a c t i n the presence of s a l t and with the previous conclusion (Section 1.4) that the COOH-terminus of P-TM can more r e a d i l y i n t e r a c t with the N^-terminus of C-TM than with the NH2"terminus of another P-TM molecule. A recent paper by P r u l i e r e et a l . (35) has shown that p i g p l a t e l e t TM has a much greater a b i l i t y to polymerize at low i o n i c strength than does horse p l a t e l e t TM and can bind to p l a t e l e t a c t i n at p h y s i o l o g i c a l s a l t conditions, 100 mM KC1 and 1-2 mM'free Mg^+ more r e a d i l y than can 71 -horse p l a t e l e t TM. Fowler and Bennett (61) have p u r i f i e d a non-muscle tropomyosin from erythrocyte membranes that also displays actin-binding properties c l o s e r to those of rabbit s k e l e t a l muscle TM than to horse p l a t e l e t or bovine b r a i n TM's (62). Like t h e i r s k e l e t a l counterparts, non-muscle TM's vary among d i f f e r e n t animal species, even though they may have derived from the same c e l l type. The f u n c t i o n a l s i g n i f i c a n c e of these v a r i a t i o n s promises to be an i n t e r e s t i n g t o p i c f o r further study. As f a r as future research with P-TM i s concerned, i t would be i n t e r e s t i n g to pursue the work on thermal denaturation using c i r c u l a r dichroism, fluorescence energy transfer and photoreactive cross - l i n k i n g agents. The presence of a reactive cysteine group at the COOH-terminus gives unique access to that area of a two chain c o i l e d c o i l p r o t e i n and the opportunity to design experiments to test the theories of Skolnick and Holtzer f o r thermal denaturation of such proteins. 2.8 Conclusions Cysteine residues at the penultimate p o s i t i o n of the COOH-terminus sequence of P-TM are capable of reaction with s u l f h y d r y l - s p e c i f i c fluorescent probes. PIA and AD were used to investigate the reduced p o l y m e r i z a b i l i t y of P-TM with respect to muscle TM's. The change of r a t i o of excimer/monomer fluorescence of Py-P-TM and the change i n p o l a r i z a t i o n and i n t e n s i t y of fluorescence from AD-P-TM i n the presence of u n l a b e l l e d TM species indicate that the COOH-terminus of P-TM can 72 -more r e a d i l y i n t e r a c t with the NH2-terminus of C-TM than with the NH2-terminus of P-TM. This, coupled with the strong homology between the COOH-terminus of P-TM and the COOH-terminus of smooth muscle TM which r e a d i l y undergoes end-to-end polymerization i n low i o n i c strength sol u t i o n s , suggests that the a l t e r e d NH2-terminus of P-TM i s the cause of i t s reduced p o l y m e r i z a b i l i t y . The emission c h a r a c t e r i s t i c s already mentioned also show that both Py-P-TM and AD-P-TM r e t a i n t h e i r a c t i n -binding c a p a b i l i t i e s , although the bulky pyrene groups may diminish the tendency of Py-P-TM to polymerize. The temperature-dependence of p o l a r i z a t i o n from AD-P-TM, AD-C-TM and AD-labelled truncated P-TM allowed construction of P e r r i n p l o t s which suggest that the COOH-terminus of P-TM does not completely unfold u n t i l ~50°C, while the Cys-190 and Cys-153 regions of AD-C-TM and AD-labelled truncated P-TM, resp e c t i v e l y , become s t e a d i l y larger loops of random c o i l . 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