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Coupled structural responses in tropomyosin Clark, Ian David 1990

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COUPLED STRUCTURAL RESPONSES IN TROPOMYOSIN By I A N D A V I D C L A R K B.Sc. Heriot-Watt University, 1984 Sc. University of B r i t i s h Columbia, 1987 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 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 OCTOBER 1990 ©Ian David Clark, 1990 i In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia/ I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis f o r sch o l a r l y purposes may be granted by the Head of my Department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. IAN D. CLARK Department of Chemistry The University of B r i t i s h Columbia, 2036 Main Mall, Vancouver, Canada V6T 1Y6 Date: October 1990 ABSTRACT Fluorescence spectroscopy can be used to probe protein conformation and i s recognized as a technique that provides very s p e c i f i c information. It has been applied/ i n recent years/ to the study of tropomyosin (TM) and i t s role i n regulation of c o n t r a c t i l e processes. In t h i s t h e s i s , two d i f f e r e n t approaches were used to further the understanding of the structure/function r e l a t i o n s h i p i n the two chain c o i l e d c o i l of tropomyosin. The f i r s t involves a comparative study on TM and non-polymerizable TM (NPTM) (Mak, A.S., and S m i l l i e , L.B. (1981) Biochim. Biophys. Res. Commun., 101, 208-214) . Fluorescence involving pyrene (Py) and acrylodan (AD) bound at the only cysteine residue i n the molecule (Cys-190), and c i r c u l a r dichroism (CD) studies led to the main conclusion that, while the two species, are very s i m i l a r i n s t a b i l i t y , the COOH-terminus i s required to hold the Cys-190 region i n a s p e c i f i c conformation. This long-range s t r u c t u r a l e f f e c t may play a role i n regulation of contraction. A species having one in t a c t COOH-terminus, made by hy b r i d i z i n g TM and NPTM, was found to be non-polymerizable suggesting that one in t a c t COOH-terminus i s i n s u f f i c i e n t to permit overlap with the NH 2-terminus of a neighbouring TM under polymerizing conditions. Unlike the TM/NPTM hybrid, the hybrid of TM and p l a t e l e t TM (P-TM) was d i f f i c u l t to make due to the sequence mismatches i n the terminal regions, i i i but small quantities could be detected by loss of excimer fluorescence from Py-P-TM on rapid cooling of a heated mixture of Py-P-TM and cardiac TM (C-TM). The second approach was to investigate the e f f e c t of actin-binding proteins on the structure and function of tropomyosin. DNase I depolymerizes F-actin and i s known to i n t e r f e r e with the end-to-end polymerizability of tropomyosin (Payne, M.R., Baydoyannis, H., and Rudnick, S.E. (1986) Biochim. Biophys. Acta 883, 454-459). Results presented here from fluorescence studies suggest that t h i s e f f e c t i s caused by a l o c a l i z e d loss of structure i n the tropomyosin at the s i t e s of l a b e l l i n g upon binding of DNase I. This r e s u l t i s supported by CD studies on l a b e l l e d and unlabelled tropomyosins. G e l s o l i n i s another actin-binding protein found i n many c e l l types and i n e x t r a c e l l u l a r f l u i d s . It i s shown here to be able to depolymerize tropomyosin, but i t s mechanism of action i s not the same as that of DNase I. The e f f e c t of in t e r a c t i o n of g e l s o l i n on the structure of tropomyosin, as determined from fluorescence studies, i s n e g l i g i b l e . TABLE OF CONTENTS i v Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1.1. PROLOGUE 1 1.2. FLUORESCENCE SPECTROSCOPY 1.2.1. P r i n c i p l e s 1 1.2.2. Chromophore Environment 3 1.2.3. Fluorescence P o l a r i z a t i o n 6 1.2.4. Excimer Fluorescence 9 1.3. CONTRACTION AND TROPOMYOSIN 1.3.1. S t r i a t e d Muscle 11 1.3.2. Regulation of Actin-Myosin Interaction 12 1.3.3. Skeletal Tropomyosin 15 1.3.4. P l a t e l e t Tropomyosin 18 1.4. PHYSICAL STUDIES OF TROPOMYOSIN 1.4.1. S t a b i l i t y Studies 22 1.4.2. Tropomyosin Hybrids 25 1.4.3. Fluorescence and Tropomyosin 28 V 2 . M A T E R I A L S A N D M E T H O D S 2 . 1 . P R O T E I N P R E P A R A T I O N 2.1.1. P u r i f i c a t i o n of Proteins 36 2.1.2. Carboxypeptidase A Treatment of C-TM ... 37 2.1.3. Fluorescent La b e l l i n g of Tropomyosins .. 39 2 . 2 . P H Y S I C A L M E T H O D S 40 3 . R E S U L T S A N D D I S C U S S I O N : A L T E R E D C O O H - T E R M I N U S O F C - T M 3 . 1 . E F F E C T O F C A R B O X Y P E P T I D A S E A 43 3.1.1. C i r c u l a r Dichroism of C-TM and NPTM .... 43 3.1.1.a. Peptide bond CD 43 3.1.1.b. Tyrosyl CD 47 3.1.2. Py-C-TM and Py-NPTM 50 3.1.3. AD-C-TM and AD-NPTM 59 3 . 2 . C - T M / N P T M H Y B R I D I Z A T I O N 62 3 . 3 . C - T M / P - T M H Y B R I D I Z A T I O N 67 4 . R E S U L T S A N D D I S C U S S I O N : A C T I N - B I N D I N G P R O T E I N S A N D T R O P O M Y O S I N 4 . 1 . I N T E R A C T I O N O F D E O X Y R I B O N U C L E A S E I W I T H T R O P O M Y O S I N 73 4.1.1. Fluorescence Studies 74 4.1.2. C i r c u l a r Dichroism 81 4 . 2 . I N T E R A C T I O N O F G E L S O L I N W I T H T R O P O M Y O S I N 84 4.2.1. V i s c o s i t y 85 4.2.2. Fluorescence Studies 86 v i 5. CONCLUSIONS 90 6. BIBLIOGRAPHY 93 7 . APPENDIX I - LIST OF SYMBOLS AND ABBREVIATIONS . . . 99 v i i LIST OF TABLES Table Page I C-TM/NPTM Hybrid V i s c o s i t i e s 63 II Excimer/Monomer Ratios for Py-P-TM/C-TM Hybrids 69 III E f f e c t of DNase I on AD-TM Fluorescence ... 75 v i i i LIST OF FIGURES FIGURE PAGE 1 Schematic diagram f o r the processes involved i n fluorescence 2 2 Fluorescent molecule acrylodan and i t s precursor, prodan 5 3 Diagrammatic representation of ex c i t a t i o n with 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 emission detection at 90° 7 4 Pyrene derivatives that react s p e c i f i c a l l y with -SH groups i n proteins 10 5 The main constituents of s k e l e t a l muscle, a c t i n and myosin 13 6 a) Thin filament constituents, actin, tropomyosin and troponin 14 b) Model for the regulation of muscle contraction 14 7 End-on view of the c o i l e d c o i l of tropomyosin from the NH 2-terminus' 15 8 Amino acid sequence of a-chain of tropomyosin 17 9 a) Alignment of sequences of S-TM and P-TM to maximize homology 20 b) E f f e c t of i o n i c strength on v i s c o s i t y of aa-TM and P-TM 20 10 E f f e c t of Mg 2 + on binding of aa-TM and P-TM to ac t i n 21 11 Representation of Cys-190 crosslinked aa-TM at various temperatures 24 12 E f f e c t of a) GuHCl and b) temperature on crosslinked and non-crosslinked TM measured by po 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 c i t y 29 13 Schematic model of the p r i n c i p a l TM conformations during unfolding 31 i x 14 Perrin p l o t s of acrylodan-labelled TMs .... 34 15 a) E f f e c t of temperature on normalized e l l i p t i c i t i e s (222nm) of C-TM and NPTM 44 b) as above f o r AD-C-TM and AD-NPTM 45 16 Time course of e f f e c t of carboxypeptidase A on e l l i p t i c i t y (222nm) of C-TM 47 17 Tyrosine CD (280nm) melting of C-TM and NPTM 4 9 18 a) E f f e c t of temperature on Py-C-TM and Py-NPTM emission c h a r a c t e r i s t i c s at high i o n i c strength 51 b) As above i n low i o n i c strength 52 19 a) Excimer/monomer r a t i o s from F i g . 18a 56 19 b) As above for non-dephosphorylated species 56 20 a) Fluorescence spectra of Py-C-TM before and a f t e r treatment with carboxypeptidase A 58 b) Decrease i n excimer fluorescence i n Py-C-TM + carboxypeptidase A 58 21 Perrin plots of AD-C-TM and AD-NPTM 59 22 Decrease i n AD-C-TM p o l a r i z a t i o n upon addition of carboxypeptidase A 61 23 Emission spectra from Py-P-TM/C-TM hybrid samples (1:1) 70 24 As above except Py-P-TM/C-TM i s 1:5 71 25 Emission spectra of AD-C-TM +/- DNase I ... 74 26 Quenching of AD-TMs +/- DNase I 76 27 P o l a r i z a t i o n changes i n AD-TMs upon addition of DNase I 78 28 Quenching of DNS-C-TM +/- DNase I 80 29 Quenching of DNase I Tryptophan fluorescence +/- tropomyosin 81 30 CD spectra of DNase I and TM 83 31 E f f e c t of g e l s o l i n on C-TM v i s c o s i t y 87 X 32 Quenching of g e l s o l i n Tryptophan fluorescence +/- C-TM 89 ACKNOWLEDGEMENTS I would l i k e to thank Dr. L e s l i e Burtnick for his guidance and support during the course of t h i s project and for h e l p f u l discussions on the content of the t h e s i s . Thanks also to Dr. L.B. S m i l l i e at the University of Alberta for providing f a c i l i t i e s f o r blood p l a t e l e t i s o l a t i o n . Special thanks to my parents for a l l t h e i r support. 1 1 . INTRODUCTION 1 . 1 . PROLOGUE The purpose of t h i s thesis was to examine s t r u c t u r a l s u b t l e t i e s of tropomyosin. Tropomyosin i s a two stranded c o i l e d c o i l protein found i n muscle and non-muscle sources. It i s e s s e n t i a l l y 100% a-helical, p r e c i p i t a t e s near i t s i s o e l e c t r i c point at pH 4.4-4.6 and i s resist a n t to denaturation at low pH and high temperature (reviewed by S m i l l i e , 1979, and C6te, 1983). 1 . 2 . FLUORESCENCE SPECTROSCOPY 1 . 2 . 1 . P r i n c i p l e s The main physical technique employed i n t h i s work i s fluorescence spectroscopy. Luminescence i s the emission of photons from e l e c t r o n i c a l l y excited states of atoms and molecules. Absorption of a photon occurs i n about 10" 1 5 seconds and i s governed by spin s e l e c t i o n rules. In the case of protein studies, since tryptophan and tyrosine ( i n t r i n s i c chromophores) and most fluorescent probes (extrinsic chromophores) have an even number of (paired) electrons, the t r a n s i t i o n s are between the ground singlet state (S0) and higher excited s i n g l e t states (S x, S2, etc.) (Figure 1). 2 Because absorption occurs on the 10~ 1 5 second timescale and the v i b r a t i o n a l motions of the chromophores are on the IO" 1 second timescale, t r a n s i t i o n s are represented as v e r t i c a l , leaving most chromophores in v i b r a t i o n a l l y excited states a f t e r absorption of a photon (Franck-Condon p r i n c i p l e ) . In solution, t h i s excess v i b r a t i o n a l energy i s dissipated Vibrational relaxation FIGURE 1. Schematic diagram f o r the processes involved i n e x c i t a t i o n and de-excitation of fluorescent molecules (from Cantor and Schimmel, 1980). rapidly to the surrounding solvent cage v i a c o l l i s i o n s , relaxing the molecule to the lowest v i b r a t i o n a l l e v e l of S x Further deactivation of the molecule back to i t s e l e c t r o n i c ground state then occurs either by emission of a photon (fluorescence, rate constant k F) 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 i c, k i s and k q / respectively) . The quantum y i e l d of fluorescence (<j>F) i s the r a t i o of the number of photons emitted to photons absorbed and can be expressed as : <|>F = k F/(k F + k i o + k i a + k q[Q]) (1.1) where [Q] i s the concentration of quencher molecules. Fluorescence i n t e n s i t y decays exponentially af t e r e x c i t a t i o n with a pulse of l i g h t so that i n t e n s i t y at time t, I t , i s given by : I t = I 0 exp(-t/x F) (1.2) where I 0 i s fluorescence i n t e n s i t y immediately aft e r the ex c i t a t i o n pulse and fluorescence l i f e t i m e : T F = (k F + k i c + k i s + k q [ Q ] ) ~ l . (1.3) 1 . 2 . 2 . Chromophore Environment Environmental factors can strongly influence the fluorescence emission of a molecule. The emission spectra of most fluorescent molecules i n solution are broad/ single peaks lacking i n v i b r a t i o n a l fine structure. This i s a r e s u l t of l i n e broadening by the solvent. 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 . It i s these variables, as well as fluorescence 4 p o l a r i z a t i o n (Section 1.4.)/ that are exploited when fluorescence i s used as a biophysical t o o l . Since the excited states of fluorescent molecules tend to be more polar than the ground states, there i s greater relaxation of the excited state i n a polar solvent than i n a non-polar solvent, leading to a decrease i n the S ^ S Q 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 water (Weber and F a r r i s , 1979). 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-ra d i a t i v e deactivation processes are more favoured i n polar solvent ( k i c and k i s increase). In the case of proteins l a b e l l e d with fluorescent probes, information on the changing p o l a r i t y of the binding s i t e , and hence changing conformation of the protein i n the binding region, can be obtained by following the eff e c t s of such variables as protein addition, temperature and i o n i c strength on the fluorescence emission maximum, in t e n s i t y and l i f e t i m e of the probe. The 6-acyl-2-dimethylaminonaphthalene moiety has been shown to emit fluorescence that i s extremely sensitive to solvent p o l a r i t y . Prendergast et al. (1983) synthesized acrylodan from prodan (Figure 2) to give a probe that reacts s p e c i f i c a l l y with t h i o l groups but s t i l l has the spectral c h a r a c t e r i s t i c s of the 6-acyl-2-dimethylaminonaphthalene group. This has made i t very useful for the study of C2H5 H ^ N PRODAN o H H / Hf ACRYLODAN FIGURE 2. T h i o l - s p e c i f i c fluorescent probe, acrylodan, and i t s precursor, prodan (Prendergast et al., 1983). conformational changes i n proteins containing reactive cysteine residues. Another exploitable property of the fluorescence emission that i s dependent on the environment of the chromophore i s that the quantum y i e l d * <}>F , can be decreased by addition of dynamic or c o l l i s i o n a l quencher molecules, Q (Equation 1.1). The bimolecular rate constant for quenching of fluorescence (k q) w i l l depend on the a c c e s s i b i l i t y of the chromophore to the quencher and any change i n t h i s i s r e f l e c t e d i n a change of slope of a plot of F 0/F vs. [Q] from the Stern-Volmer equation: F 0/F = 1 + k q T 0 [Q] (1.3) 6 where F 0 i s fluorescence i n the absence of quencher, x0 i s the l i f e t i m e of the excited state i n the absence of quencher and k q i s the bimolecular rate constant for quenching. One of the best known c o l l i s i o n a l quenchers i s molecular oxygen. Its concentration i n a given protein solution may be taken to be constant, so i t may be ignored. There are many species that are commonly used as quenchers, for example, Xe, H 20 2, acrylamide, N20, Cs + and I". The iodide ion works well with acrylodan (Clark, 1987) and i s thought to induce an intersystem crossing i n the chromophore to an excited t r i p l e t state v i a spin-orbit coupling (Kaska, 1952). 1.2.3. Fluorescence P o l a r i z a t i o n If plane polarized l i g h t i s used to excite a chromophore on a macromolecule i n solution, the fluorescence observed at 90° to the incident beam w i l l be polarized to a degree that depends on the size, 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 arrangement for p o l a r i z a t i o n measurements, \i i s the absorption dipole moment of the fluorescent species. The a b i l i t y of \l to interact with the v e r t i c a l o s c i l l a t o r f a l l s o f f with c o s 2 9 , producing a set of excited molecules that i s symmetrical about the z-axis. 7 Z A Incident light Detector 4 A X FIGURE 3. Diagrammatic representation of excitation of a chromophore with v e r t i c a l l y p o l a r i z e d l i g h t . P o l a r i z a t i o n (p) and anisotropy (A) are defined: where I ^  and Ij^, respectively, are inte n s i t y of emission p a r a l l e l and perpendicular to the plane of exc 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 . P o l a r i z a t i o n , p, was introduced as an analogue to the dichroic r a t i o , d, that i s used to quantitate l i n e a r dichroism of a substance (1.4) (1.5) 8 [d = (Abs. - Abs. )/ (Abs. + Abs. ) ] . However, due to the spherical d i s t r i b u t i o n of fluorescence emission, the term (I + 21 ) i s d i r e c t l y related to the t o t a l emission i n t e n s i t y , a fact that makes anisotropy values more convenient to work with i n further t h e o r e t i c a l developments, s p e c i f i c a l l y i n time-dependent studies of anisotropy decay. For steady-state fluorescence measurements, which involve an averaging of emission p o l a r i z a t i o n , the p term i s adequate i n most cases for access to information on probe motions and macromolecular f l e x i b i l i t y . An expression that relates fluorescence parameters p and X to factors that a f f e c t molecular rotation rates for spheres i n solution, v i s c o s i t y (Tj), temperature (T), and molecular volume (V) i s the Perrin equation: (1/p - 1/3) = ( l / p 0 - 1/3) (1 + TkT/VTj) (1.6) The P e r r i n plo t i s a plo t of (1/p - 1/3) vs. T / T J with a slope of ( l / p 0 - 1/3) (xk/V) and an intercept of ( l / p Q - 1/3). The V term becomes more complex for aspherical molecules because r o t a t i o n a l d i f f u s i o n i n three dimensions must be considered. The term p 0 i s the i n t r i n s i c fluorescence p o l a r i z a t i o n . This i s the degree of p o l a r i z a t i o n of the fluorophore i f i t i s held r i g i d l y during the excitation/emission process and a l l other e x t r i n s i c depolarizing factors (light scattering, reabsorption and emission) are absent. In the case where the absorption and emission dipoles are p a r a l l e l ( i . e . the angle, P, between 9 the dipoles i s zero), then p 0 = 0.5. The general expression r e l a t i n g p 0 and B i s : p 0 = (3cos 2 p - l ) / ( 3 + cos 2 B) ( 1 . 7 ) For most fluorescent molecules/ the symmetry of the excited state i s the same as that of the ground state and B = 0 . Therefore, p o l a r i z a t i o n measured for these molecules can t h e o r e t i c a l l y have values between 0.5 and 0, depending on the r o t a t i o n a l mobility and fluorescence l i f e t i m e of the molecule. 1 . 2 . 4 . Excimer Fluorescence The term excimer i s short for excited dimer and i s used to describe the dimer formed on i n t e r a c t i o n of two i d e n t i c a l solute molecules/ one e l e c t r o n i c a l l y excited and the other i n the ground state. Excimers can fluoresce and exhibit a broad, structureless emission at longer wavelengths than observed f o r the excited monomer. The most commonly studied excimer-forming species i s pyrene ( t h i o l - s p e c i f i c derivatives shown i n Figure 4). Pyrene has a r e l a t i v e l y long fluorescence l i f e t i m e which allows an excited molecule time to form a face-to-face sandwich arrangement with a nearby ground state molecule. This i s an a t t r a c t i v e i n t e r a c t i o n with an energy minimum at a pyrene-pyrene separation of about 4A. Pyrene monomer emission has some v i b r a t i o n a l fine structure. However/ there i s none i n the excimer emission because no v i b r a t i o n a l l e v e l s are available due to a repulsive i n t e r a c t i o n that forbids ground state dimer formation. N-(l-pyrene)maleimide (PM) N-(1-pyrene)iodoacetamide (PIA) F I G U R E 4. T h i o l - s p e c i f i c derivatives of pyrene. Since the formation of an excimer requires there to be two pyrene molecules close enough together to interact before the excited monomer emits, t h i s e f f e c t i s obviously concentration dependent for free pyrene molecules i n s o l u t i o n . For pyrene i n ethanol/ a pyrene concentration of >10"4M i s needed before appreciable excimer formation can be detected. However, i f two pyrene molecules can be f i x e d i n close proximity to each other on a polymer chain or on a surface, then the need for d i f f u s i o n p r i o r to i n t e r a c t i o n i s 11 removed. Such a s i t u a t i o n occurs i n pyrene-labelled tropomyosin, f i r s t observed by Ohyashiki et al. (1976). 1.3. CONTRACTION AND TROPOMYOSIN 1.3.1. S t r i a t e d 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 involving mechanical stress, t o r s i o n and translocation. The knowledge that has been obtained concerning the structure and function of a c t i n and myosin comes largely from studies on s t r i a t e d muscle. S t r i a t e d muscle i s a highly s p e c i a l i z e d t i s s u e 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 . For t h i s purpose i t contains large amounts of a c t i n and myosin organized into c l o s e l y packed, highly ordered arrays (Figure 5). Myosin i s aggregated to form bipolar thick filaments which alternate between the t h i n filaments of a c t i n that are anchored to perpendicular l i n e s of protein c a l l e d a -actinin ( Z - l i n e s ) . The t h i n filament also contains the regulatory proteins troponin and tropomyosin. The precise molecular mechanism of the actin-myosin in t e r a c t i o n i s s t i l l the 12 subject of debate but the generally accepted model for contraction i s that f i r s t proposed by Huxley (1969) on the basis of electron 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, translocating and releasing, the ac t i n to p u l l adjacent Z-lines closer together, shortening the muscle (Figure 5). 1.3.2. Regulation of Actin-Myosin Interaction The i n t e r a c t i o n of act i n and myosin i s controlled, i n s t r i a t e d 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 + concentration, either allow or prevent myosin heads from binding to a c t i n . A c t i n filaments are made up of in 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 stoichiometry 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, respectively. Potter and Gergely (1974) produced a model for the calcium-dependence of muscle contraction (Figure 6b). Upon addition of Ca 2 + and subsequent binding to TN-C, a conformational change occurs i n which the tropomyosin s l i d e s deeper into the groove of the ac t i n double h e l i x allowing myosin access to the ac t i n filament. 13 /H Z A I s'band line band bond ; Mvofiliril Z urcomcre Z / \ / V / / A band \ I -f—ii * , r V l TT/ ir I z m m n u n m m n u n n u n m m H I H I j i n n / lamer osin| .\ . H itn H U H i H U H \ Z n u n i in in i n u n i n u n i l l l l l l i H U H Thick fiUments (myosin) I m i n IUIII H I H I HIIII n u n m m HIIII H I H I 7 Thin filaments (actin) Cross sections at points indicated FIGURE 5. The main constituents of sk e l e t a l muscle, act i n and myosin (from Lehninger, 1975) . 14 FIGURE 6 a) Thin filament constituents, actin, tropomyosin and troponin (TN-I, TN-C and TN-T) (From C6te, 1983). b) Model for the regulation by [ C a 2 + ] . i) pCa 2 + = 8. i i ) pCa 2 + = 5. (from Potter and Gergely, 1974) . 15 1.3.3. Skel e t a l Tropomyosin Tropomyosin was f i r s t i s o l a t e d i n 194 6 from rabbit s k e l e t a l muscle (Bailey, 1946). As depicted i n Figure 6a, tropomyosin i s a rod-like protein 42nm i n length. It has a molecular weight of 66,000 and i s composed of two highly h e l i c a l subunits (>95%) wrapped around each" other to form a c o i l e d c o i l . The two strands run p a r a l l e l to each other and are i n r e g i s t e r (Lehrer, 1975). Since two turns of an a-h e l i x contain seven amino acid residues, a two stranded a-h e l i c a l c o i l e d c o i l , represented end on i n Figure 7, can be viewed as a pseudo-repeating heptapeptide that i s s t a b i l i z e d by interactions between hydrophobic residues at positions 2 and 5 i n the heptapeptide sequence. FIGURE 7. Cross-section of the c o i l e d c o i l of tropomyosin looking from the NH 2-terminus end (from Hodges et a l . , 1981) . 16 The sequence of rabbit s k e l e t a l tropomyosin clo s e l y follows t h i s pseudo-repeating pattern through i t s whole length (Figure 8). A high proportion of a c i d i c and basic residues are found paired i n positions 6 and 1 of adjacent chains 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 s . Rabbit s k e l e t a l tropomyosin contains two types of polypeptide chain, a and P, which are present i n a r a t i o of approximately 4:1. Both forms are 284 residues long but show 39 differences i n t h e i r amino acid sequences(Hodges et al., 1972; Stone et al., 1975; Stone and Sm i l l i e , 1978). The a subunit migrates somewhat more rapidly on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Important to note i s the presence of cysteine residues at positions 190 of both a and P chains and at po s i t i o n 36 of the P chain. In muscle, tropomyosin binds head-to-tail along the th i n filament (Figure 6a). This head-to-tail interaction i s responsible for i t s polymerization i n low i o n i c strength solutions, as observed by v i s c o s i t y measurements (Kay and Bailey, 1960) and for i t s cooperative binding to acti n (Yang et al., 1979; Wegner, 1979). The int e r a c t i o n involves overlap of 8 to 9 residues of the COOH- and NH 2-termini of adjacent tropomyosin molecules. When residues 274-284 were removed from the COOH-terminus of rabbit cardiac tropomyosin (Ueno et al., 1976; Mak and Sm i l l i e , 1981a), i t s a b i l i t i e s to polymerize and to bind to F-actin were l o s t . Several studies have shown that troponin, e s p e c i a l l y the Tn-T 17 1 AcMet 14 Asp -28 Asp -42 Glu -56 Glu -70 Lys -84 Asp — 98 Glu -112 Lys — 126 Gly -140 Lys -154 H e -168 Lys -182 Arg -196 Glu -210 Gin -224 Glu -238 Arg -252 Ser -266 Lys -280 Asp -15 Lys 2 -Asp 16 Glu 29 Lys -Lys -43 Leu -Val -57 Leu -Asp -71 85 99 30 44 58 72 86 100 113 Leu 114 f-Glu 127 Met 141 Met 128 -Lys 142 K l u 155 Ala 156 r-Glu 169 Leu 183 Ala 197 Leu 211 Ala 225 239 Ala 170 •Val 184 r-Glu 198 •Lys 212 r-Glu 226 240 Glu 253 H e 267 281 Met 254 Asp 268 -Lys 282 Thr 3 ( ^ \ 5 Ala -\Iley-Lys 17 ( 18\ 19 Asn AAla/-Leu 31 ^32\ " 33 Ala AAla/-Glu 45 /V\ 47 Ser \Leu/-Gln Leu -Glu -Val -Ala -Leu -Asp — HeJ-Lys -6 7 Lys -Lys 20 21 Asp -Arg 34 35 Asp -Arg 48 49 Lys -Lys 62 63 Glu -Ala 76 77 Lys -Lys 90 91 Arg -Arg 104 105 Glu -Arg 118 119 Lys -Ala 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 13 Leu 27 Ala 41 Asp 55 Asp 69 Glu 83 Ala 97 Glu 111 Gin 125 Arg 139 Glu 153 His 167 Arg 181 Glu 195 Glu 209 Ala 223 Glu 237 Thr 251 Lys 265 Leu 279 Asa FIGURE 8 Amino a c i d s e q u e n c e o f a - c h a i n o f t r o p o m y o s i n . I d e a l i z e d n o n - p o l a r r e s i d u e p o s i t i o n s a r e shown i n s q u a r e s a n d c i r c l e s ( f r o m S t o n e et al., 1 9 7 5 ) . 18 component, can restore the a b i l i t y of t h i s nonpolymerizable tropomyosin (NPTM) to bind to F-actin v i a bridging over the deleted overlap region i n NPTM (Mak et a l . , 1983; Heeley et al. 1987) . However, the t h i n filament reconstituted with NPTM shows s i g n i f i c a n t l y reduced ac t i v a t i o n of the Mg 2 +-ATPase a c t i v i t y of bound myosin subfragment 1 i n the presence of C a 2 + r e l a t i v e to the system with in t a c t TM (Heeley et al., 1989). This emphasizes the significance of the overlap region of TM i n regulating the function of the t h i n filament. 1.3 . 4 . P l a t e l e t Tropomyosin It 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 involving movement. Microfilaments, which are e s s e n t i a l l y F-actin, are the most studied component of non-muscle force generators. They interact with many a c t i n -binding proteins, whose a c t i v i t i e s i n turn, are modulated by non-muscle tropomyosins bound to the a c t i n (Payne and Rudnick, 1984). 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 (Cohen and Cohen, 1972). It was recognized as a tropomyosin by i t s >95% h e l i c a l structure, p r e c i p i t a t i o n at pH 4.6 and the s i m i l a r i t y of i t s amino acid composition to that of the a chain of TM. The main feature that distinguishes i t and most other non-muscle TMs from muscle TM i s t h e i r smaller size, 19 the subunit molecular masses being 26-30,000 daltons and 33,000 daltons, respectively (C6te, 1983). Equine p l a t e l e t TM (P-TM) contains 247 residues i n each subunit, with cysteines at positions 153 and 246 (Lewis et al., 1983). As the r a t i o of B/a i n P-TM i s greater than 2/1, the amino acid sequence obtained i s most l i k e l y that of the B 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 clear 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. In segments extending from residues 81 to 258 of cc-TM (44 to 221 of P-TM), no insertions or deletions occur. However, immediately to the COOH- and NH 2-terminal sides of t h i s section s i g n i f i c a n t differences between the sequences of the two proteins occur. These observations are explained by a l t e r n a t i v e s p l i c i n g of exons within single TM genes that have been shown to be capable of producing both smooth and s k e l e t a l a-TM (Ruiz-Opazo and Ginard, 1987) or s k e l e t a l B-TM plus f i b r o b l a s t (non-muscle) TM (Helfman et al., 1986). In a l i g n i n g the sequences of the muscle and p l a t e l e t TMs to maximize homology (Figure 9a), i t can be seen that the lower molecular weight of P-TM i s accounted for by two 21 residue deletions within the f i r s t 80 residues of the muscle TM. It should also be noted that the NH 2-terminus of P-TM i s extended by 5 residues. Although these alterations do not seem to disrupt the a-helical structure of the 20 p r o t e i n , they profoundly a f f e c t P-TM's a b i l i t y to function i n the same way as does muscle TM in vitro. The head-to-tail i n t e r a c t i o n of P-TM i n low i o n i c strength solutions i s much weaker than that of aa-TM, as can be seen from the v i s c o s i t y data (Figure 9b) (C6te et al., 1978). a) KCI (M) FIGURE 9. a) Alignment of sequences of S-TM and P-TM to maximize homology. Speckled region - sequences very si m i l a r ; cross-hatched region -sequences d i f f e r e n t ; blank region -areas of S-TM which are deleted i n P-TM (From C6te, 1983) . b) Relative v i s c o s i t y vs. i o n i c strength rel a t i o n s h i p for s k e l e t a l oca-TM (•» and P-TM (O) (from C6te et al., 1978). 21 Stoichiometric binding of P-TM to actin, as measured by a sedimentation assay, requires Mg 2 + concentration of 6-8mM, as opposed to 2mM for aa-TM (Figure 10) (C6te and S m i l l i e , 1981a). This, along with the fact that there i s a higher temperature in vivo (37°C vs. 20°C i n the binding study) and that free cytoplasmic Mg 2 + concentration i s about ImM, suggests that equine P-TM would bind poorly to free a c t i n filaments in vivo. However, recent studies on p i g P-TM MgCl2 (mM) F I G U R E 10. Effect of increasing amounts of Mg 2 + on the binding of skeletal aa-TM (•) and P-TM (•, A) to ac t i n (from C6te and Smillie, 1981). (Pruliere et a l . , 1986) have shown that binding to a c t i n i s >90% complete under physiological conditions (lOOmM KC1, 1-2mM Mg 2 +), suggesting that there could be a s i g n i f i c a n t 22 functional difference between P-TMs from d i f f e r e n t animal species. 1 . 4 . PHYSICAL STUDIES OF TROPOMYOSIN 1 . 4 . 1 . S t a b i l i t y Studies The fact that i o n i c strength a f f e c t s the end-to-end poly m e r i z a b i l i t y of TM suggests that i t i s the r e s u l t of i o n i c and/or polar interactions between adjacent molecules. In contrast/ however, the s t a b i l i t y of i n d i v i d u a l molecules i s due primarily to hydrophobic i n t e r a c t i o n between the chains, so increased i o n i c strength has a s t a b i l i z i n g e f f e c t on i n d i v i d u a l TM molecules. This i s borne out by thermal denaturation studies (Betteridge and Lehrer, 1983; I s h i i and Lehrer, 1986) that follow the loss of cc-helical structure by c i r c u l a r dichroism. 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/ aa-TM) occurs around 50°C i n 0.5M NaCl and at 38°C i n 4mM NaCl. At high i o n i c strength, oxidized forms of both cardiac and s k e l e t a l TM show a small loss of h e l i x at 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 provided by fluorescence and c i r c u l a r dichroism (Lehrer, 1978), NMR (Edwards and Sykes, 1980), ESR (Graceffa and Lehrer, 1984) and d i f f e r e n t i a l scanning calorimetry (Williams and Swenson, 1981). 23 Because TM i s almost e n t i r e l y a-helical, i t has been used as an experimental model i n the development of theories concerning a-helix to random c o i l t r a n s i t i o n s i n two chain c o i l e d c o i l s (Skolnick and Holtzer, 1985; 1986) . Using predetermined values for h e l i x i n i t i a t i o n and propagation for each amino ac 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, Skolnick and Holtzer computed t h e o r e t i c a l % h e l i x vs. temperature plots for reduced aa-TM and f o r aa-TM crosslinked 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 dichroism. The large p r e t r a n s i t i o n present i n TM chains crosslinked at Cys-190 was proposed by Lehrer (1978) to be due to the fact that i n the undisturbed native double hel i x , the two sulfhydryls of the Cys-190s, each of which i s at 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 bond introduces a considerable s t e r i c s t r a i n i n the c o i l e d c o i l and leads to l o c a l i z e d unfolding of the c o i l i n the Cys-190 region. The molecule at about 35°C may be pictured to be double h e l i c a l at both ends, but containing a "bubble" of random c o i l centred at the crosslink (Betteridge and Lehrer, 1983; Graceffa and Lehrer, 1980). 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 entropic cost of generating such a bubble i n the centre of a c o i l e d c o i l structure. 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 predict that, instead of forming a bubble, the molecule should unfold from the COOH-terminus to the cro s s l i n k (Figure 11). Studies on P-TM (Clark and Burtnick, 1988) support Lehrer's conclusion as do the NMR studies of Edwards and Sykes (1980) but the issue i s s t i l l not resolved. FIGURE 11. Representation of Cys-190 crosslinked oca-TM calcula 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 NH 2-terminus side and 21 residues to the COOH-terminus 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% heli x (from Skolnick and Holtzer, 1986) . 25 What i s generally agreed i s that the NH 2-terminal half of the molecule i s considerably more stable than the COOH-terminal h a l f (Woods, 1977; Pato et a l . , 1981), due to stronger i n t e r h e l i x i n t e r a c t i o n (Skolnick and Holtzer, 1983) suggesting that just as the NH 2-terminal region i s the f i n a l region to unfold during thermal denaturation, i t i s also the f i r s t region to renature upon cooling. 1 . 4 . 2 . Tropomyosin Hybrids Tropomyosin i s o l a t e d from rabbit heart muscle i s a homodimeric species (aa-TM) (Lewis and Sm i l l i e , 1980). However, rabbit s k e l e t a l muscle TM contains both aa-TM and aB-TM (a/p = 4/1) with the ap heterodimer being present at le v e l s higher than expected from random association (Bronson and Schachat, 1982). This, along with the fact that PP-TM i s not found, although r e a d i l y made in vitro, suggests that the ap-TM i s being assembled p r e f e r e n t i a l l y in vivo. Holtzer et al. (1984) showed that mixing aa-TM and PP-TM (formed separately i n solutions containing p u r i f i e d a orPchains) at room temperature f o r an extended period d i d not produce any hybrid (aP) species. Holtzer et al. (1984) also showed that the f r a c t i o n of hybrid produced on recooling heat-denatured tropomyosin solutions (0.5) was independent of (a) the temperature at which denaturation occurred, (b) the rate of cooling and (c) the f i n a l temperature of the renatured sample. The data indicated random reassociation of the tropomyosin chains ( i . e . aa:aB:BB = 1:2:1). Brown and Schachat (1985) found a s i m i l a r homodimer/heterodimer d i s t r i b u t i o n a f t e r denaturation of p u r i f i e d aB-TM with urea and subsequent renaturation by rapid d i l u t i o n . However, they found the f i n a l d i s t r i b u t i o n to be temperature dependent and found that cooling of thermally denatured tropomyosin or d i a l y s i s to remove urea from chemically denatured tropomyosin l e d p r e f e r e n t i a l l y to homodimers. The difference i n r e s u l t s may be due to the d i f f e r e n t choice of s t a r t i n g species, but has not yet been explained s a t i s f a c t o r i l y . Studies on smooth muscle TM have contributed somewhat to the understanding of the r e l a t i v e s t a b i l i t i e s of heterodimers and homodimers. Chicken gizzard TM (G-TM) i s composed of two subunits, a and B, which are present i n about equal amounts (Cummins and Perry, 1973) and d i f f e r s l i g h t l y i n amino acid sequence (Lau et a l . , 1985; Sanders and S m i l l i e , 1985). The a chain comes from the same gene that produces the sk e l e t a l a-TM, the two proteins representing products of alternative s p l i c i n g of the gene (Ruiz-Opazo and Nadal-Ginard, 1987). The a-G-TM chain retains the cysteine at po s i t i o n 190. The smooth muscle B chain has a cysteine at po s i t i o n 36 only, unlike s k e l e t a l B-TM. Sanders et al. (1986) showed that native G-TM i s predominantly aB-TM. However, when G-TM i s denatured with guanidinium HC1 then renatured, the tendency i s to form homodimers (Graceffa, 1989), suggesting that when aB-TM i s made in vivo i t i s the k i n e t i c a l l y trapped product. More 27 recently, Lehrer (1990) confirmed the resu l t s of the previous study but, by expanding the study, was able to of f e r an alt e r n a t i v e explanation. He found that upon cooling thermally denatured G-TM, BB-TM forms f i r s t ' . This then requires that the remaining a chains renature to aa-TM. If the homodimer mixture i s equ i l i b r a t e d at 37-40°C, the native aB-TM i s produced v i a chain exchange. Thus the opposite conclusion i s reached compared to the Graceffa (1989) study - the homodimers are the k i n e t i c products and the native heterodimer i s the thermodynamic product. As mentioned i n section 1.3.4., there i s a s i g n i f i c a n t portion of C-TM that i s conserved i n the P-TM sequence. This suggests that the p o s s i b i l i t y of forming a hybrid TM containing one chain of each type exists, but i s not a foregone conclusion due to the difference i n length (37 residues) and presence of regions where the sequences d i f f e r (Figure 9a), although the pseudo-repeating heptad pattern i s maintained. This i s investigated i n Chapter 3 together with hyb r i d i z a t i o n of C-TM and nonpolymerizable C-TM (NPTM). The only reported attempt to hybridize two chain c o i l e d c o i l s from d i f f e r e n t sources was with paramyosin, an invertebrate muscle protein s t r u c t u r a l l y s i m i l a r to TM but three times as long (Crimmins and Holtzer, 1981). They found that af t e r thermal denaturation and rapid cooling of paramyosins from clam and worm, the f r a c t i o n of renatured molecules that were hybrids varied from 10% at 5°C to 5% at 25°C. The equilibrium value, calculated a f t e r slow cooling, was about 28 4% and showed a small but s i g n i f i c a n t increase with increased temperature. The primary sequences of the proteins were not known so the degree of homology between the proteins could not be assessed. 1 . 4.3. Fluorescence and Tropomyosin There are six tyrosine residues and no tryptophans per a-chain of TM and t h i s was u t i l i z e d by Lehrer (1978) to study the e f f e c t on TM structure of oxidation of the -SH groups to S-S inter-chain crosslinks at Cys-190. Tyrosine has a low quantum y i e l d but s u f f i c i e n t fluorescence was produced to allow measurement of i t s i n t e n s i t y and p o l a r i z a t i o n . The fluorescence p o l a r i z a t i o n of both reduced and oxidized TMs responded to guanidine hydrochloride (GuHCl)-induced unfolding and temperature-induced unfolding i n a very s i m i l a r way to the response of the c i r c u l a r dichroism (CD) measurements at 222nm (Figure 12). The non-crosslinked TM (dashed lines) unfolds at a lower temperature than crosslinked TM ( s o l i d l i n e s ) , although the crosslinked species shows a low temperature i n s t a b i l i t y (pretransition) that Lehrer assigned to p a r t i a l unfolding i n the Cys-190 region. It i s i n t e r e s t i n g to see the tyrosine p o l a r i z a t i o n data agree so well with the CD data since f i v e of the six tyrosines are i n the COOH-terminal h a l f of the molecule while the CD signal i s averaged over the whole molecule. 29 Ohyashiki et al. (1976) u t i l i z e d the reactivity of the -SH groups in ap-TM by specifically labelling them with the fluorescent probes N-(l-anilinonaphthyl-4)maleimide (ANM). They showed that as the low ionic strength end-to-end overlap of ANM-TM was removed by addition of KC1, the p i I i i . i . i 20 40 60 80 Temperolure {'O FIGURE 12. Effect of a) guanidine hydrochloride (GuHCl) and b) temperature, on crosslinked TM ( ) and non-crosslinked TM ( ). Top: change in polarization (p). Middle: change in fluorescence intensity (F). Bottom: change in relative e l l i p t i c i t y ([6]/[8]0) at 222nm (from Lehrer, 1978). 30 fluorescence emission maximum was blue s h i f t e d and the p o l a r i z a t i o n increased. This suggested that i n monomeric TM, the conformation i s such that the probes are buried deeper i n the hydrophobic core of the molecule compared to when the TM i s polymerized. The same s h i f t s were observed upon binding of ANM-TM to F-actin from which i t was concluded that the ac t i n was inducing the same kind of conformational -change as just mentioned. The fact that the two chains of TM are capable of being crosslinked at Cys-190 made them p o t e n t i a l l y able to produce excimer fluorescence i f s u l f h y d r y l - s p e c i f i c pyrene derivatives were attached to Cys-190 residues on neighbouring chains. This was r e a l i z e d by Betcher-Lange and Lehrer (1978) who l a b e l l e d aa-TM with N- (1-pyrene)maleimide (PM) and found that the addition of GuHCl destroyed excimer fluorescence p r i o r to the main loss of secondary structure, suggesting l o c a l chain separation i n the Cys-190 region. The presence of appreciable excimer below the pr e t r a n s i t i o n temperature suggests that, since pyrenes cannot stack around the outside of the molecule, pyrene-pyrene hydrophobic interactions can open the chain around the Cys-190. An equilibrium between chain-open and chain-closed conformations i n the native protein was proposed by I s h i i and Lehrer (1985). Lehrer also showed that the introduction of pyrene moieties at Cys-190 of TM strongly i n h i b i t s low s a l t polymerization, l a t e r attributed to the 10% loss of a -h e l i x structure produced upon l a b e l l i n g ( I s h i i and Lehrer, 1986). This i s su r p r i s i n g since the COOH-terminus i s 94 residues away and suggests that the pyrenes cause a conformational change that can be transmitted over a long range. (This connection between the COOH-terminus and the Cys-190 region i s investigated i n Chapter 3 ) . I s h i i and Lehrer (1985) proposed an equilibrium scheme that explains the emission of both monomer and excimer fluorescence from pyrene-TM and how excimer fluorescence increases by means of a temperature-dependent s h i f t i n equilibrium from a chain closed state (N) to a chain open state (X) associated with the p r e t r a n s i t i o n (Figure 13 ) . M N X mums rnmwt D FIGURE 13. Schematic model which assumes two probe conformations, M and E, are the p r i n c i p a l TM conformations on the unfolding pathway. N i s the native, chain-closed, f u l l y h e l i c a l state observed at low temperature; X i s the chain-open, p a r t i a l l y unfolded intermediate; D i s the f u l l y unfolded, dissociated state (from I s h i i and Lehrer, 1985). 32 They showed that the presence of the lab e l causes a decrease i n TM binding to F-actin, possibly the re s u l t of reduced end-to-end in t e r a c t i o n s . When pyrene-TM was bound to F-actin, excimer fluorescence d i d not increase with temperature, i n d i c a t i n g that F-actin s t a b i l i z e s TM by i n h i b i t i n g the N to X t r a n s i t i o n . Further s t a b i l i z a t i o n of TM was also observed with binding 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 . < 1/7), 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 po s i t i o n of TM i n the t h i n filament. Burtnick et al. (1986) used N- (1-pyrenyl)-iodoacetamide (Py) to la 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 intera c t with other TM species and i t s a c t i n binding properties. Labelling under mild conditions gave a high degree of l a b e l l i n g (1.1 Py per chain) because 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 i n muscle TM, but rather Cys-246, the penultimate COOH-terminus residue. Excimer fluorescence dominated the emission spectrum of Py-P-TM suggesting considerable f l e x i b i l i t y of the in d i v i d u a l chains at the'COOH-terminus. They also found that the COOH-terminus of P-TM more readi l y interacts with the NH 2-terminus of C-TM than with the NH2-terminus of P-TM. This, coupled with fact that the COOH-terminal sequences of P-TM and smooth muscle TM are highly homologous (Sanders and Sm i l l i e , 1985), suggests that i t i s 33 the a l t e r e d NH 2-terminus of P-TM that i s the cause of i t s reduced a b i l i t y to self-polymerize. Burtnick et al. (1988) used the absence of excimer from l a b e l l e d native gizzard TM to provide evidence that the native species was a heterodimer, one chain having a cysteine at p o s i t i o n 36 and the other chain having a cysteine at p o s i t i o n 190. This s p a t i a l separation i s f a r beyond the 4A pyrene-pyrene distance required for excimer formation. Acrylodan-labelled TM species were used to investigate the thermal denaturation of TM (Figure 14) (Clark and Burtnick, 1988). The higher the 1/p value at a given temperature i n the Perrin plot, the more rapidly the protein-bound probe i s tumbling i n solution. The Perrin p l o t suggested that the acrylodan on Cys-24 6 of P-TM was i n a region that remained r e l a t i v e l y stable to thermal denaturation u n t i l 55°C. Figure 14 also shows that the Cys-190 region of C-TM and the analogous Cys-153 region of truncated P-TM respond s i m i l a r l y to temperature. The data suggest that these regions uncoil at a temperature below that of the main unfolding t r a n s i t i o n , consistent with the model of Graceffa and Lehrer (1980) that proposes a "bubble" of random c o i l i n the Cys-190 region that expands out towards the COOH-terminus with increasing temperature. Lehrer and I s h i i (1988) used acrylodan l a b e l l e d C-TM with F-actin and myosin SI heads to show that there i s a change i n the s p a t i a l r e l a t i o n s h i p between TM and actin that 34 8 7 6 5 Q . i 4 3 2 I 3 5 7 9 T/77 (K/poise)xlO-4 FIGURE 14. Perrin plot f o r AD-P-TM (•) , AD-C-TM (4» and AD-la b e l l e d truncated P-TM (•) (from Clark and Burtnick, 1988) . i s induced by the binding of SI i n the absence of ATP. This was concluded from observed increased energy transfer from tryptophan residues i n a c t i n to acrylodan oh C-TM. The e f f e c t was temporarily rever s i b l e i f ATP was added. Once a l l the ATP was hydrolysed by the actomyosin ATPase a c t i v i t y , the SI heads became immobilized on the actin filament. 35 The f i r s t fluorescence evidence for the in t e r a c t i o n of the actin-binding and depolymerizing protein, DNase I, with tropomyosin (Payne at a l . , 1986) came from Burtnick and Racic (1988), using a s u l f h y d r y l - s p e c i f i c coumarin reagent (DCIA) to la b e l cardiac TM. The binding of DNase I did not a l t e r the emission i n t e n s i t y or wavelength maximum but did s i g n i f i c a n t l y reduce the p o l a r i z a t i o n from 0.38 to 0.26, i n d i c a t i v e of a loosening of the c o i l e d c o i l structure i n the Cys-190 region. Stronger evidence for t h i s conclusion comes from work presented i n Chapter 4 using acrylodan-l a b e l l e d TM species (Clark and Burtnick, 1989). Also examined i n Chapter 4 i s the in t e r a c t i o n of TM with another actin-binding and depolymerizing protein, g e l s o l i n . 36 2 . MATERIALS AND METHODS 2 . 1 . PROTEIN PREPARATION 2 . 1 . 1 . P u r i f i c a t i o n of Proteins P l a t e l e t tropomyosin was p u r i f i e d from l y o p h i l i z e d horse blood p l a t e l e t s according to method 2 of C6te and Sm i l l i e (1981b). The basis of the p u r i f i c a t i o n i s the r e p e t i t i v e i s o e l e c t r i c p r e c i p i t a t i o n (pH 4.4) and resuspension (pH 7.0) of the tropomyosin with each pH change followed by centrifugation to remove the unwanted p e l l e t or supernatant. The f i n a l step was hydroxylapatite chromatography using a 0 to 0.2M phosphate gradient and pur i t y was judged to be greater than 95% by polyacrylamide gel electrophoresis (SDS-PAGE) (Laemlli, 1970). Cardiac tropomyosin was p u r i f i e d by Jane Ki s h i i n our laboratory, as outlined by S m i l l i e (1982), from acetone muscle powders of rabbit hearts prepared by several ethanol and acetone extractions of homogenized t i s s u e . G e l s o l i n was p u r i f i e d by Scott Reid i n our laboratory from horse blood plasma using the modified method of Bryan (1988) as adapted by Ruiz S i l v a and Burtnick (1990). 37 2 . 1 . 2 . Carboxypeptidase A Treatment of C-TM Cardiac tropomyosin was i n i t i a l l y dephosphorylated using Escherichia Coli a l k a l i n e phosphatase (Worthington) (Lewis and S m i l l i e , 1980; Heeley et al., 1987). C-TM (50mg) and a l k a l i n e phosphatase were dialysed overnight against 150mM KC1, 50mM T r i s , lOmM MgCl 2/ pH 8.0. The C-TM and alk a l i n e phosphatase were then incubated at 37°C for 2 hours at a r a t i o of 50:1 (w/w). Alkaline phosphatase was removed from the tropomyosin by hydroxylapatite chromatography (Sigma) (Watson et al., 1988). Dephosphorylated C-TM was then treated with DFP-treated carboxypeptidase A (Sigma) (Ueno et al., 1976; Mak and Smi l l i e , 1981). The enzyme was dissolved (2mg/ml) i n 2M NH4HC03, pH 8.0, and treated with 100^ .1 of a lmg/ml solution of phenylmethylsulfonyl chloride (PMSF) i n methanol. C-TM i n 150mM KC1, lOmM T r i s , 0.01% azide, pH 8.0, was then incubated with carboxypeptidase A i n a r a t i o of 50:1 (w/w) at 37°C for 3 hours, which can remove up to 11 residues from the COOH-terminus of C-TM. The sample was then heated to 85°C to denature the carboxypeptidase A which was removed, a f t e r cooling, by centrifugation at 15,000 xg for 10 minutes. In order to check that the enzyme was removing the COOH-terminus residues, 20u,l of the non-polymerizable tropomyosin (NPTM) solution was reacted with 20(Xl of lOmM dansyl chloride (Sigma) i n acetone and subjected to TLC on 38 polyamide sheets (Woods and Wang, 1967) i n 3 solvent systems sequentially: 1. Formic acid i n water (1.5% v/v). 2. 90° rotation, then Benzene:AcOH (9:1) 3. Ethyl acetate:AcOH:MeOH (20:1:1) The dansyl amino acids were then i d e n t i f i e d by comparison of t h e i r m o b i l i t i e s i n d i f f e r e n t solvent systems with those of standard dansyl-amino acids (Sigma) (Perham, 1978). In agreement with Mak and S m i l l i e (1'981), who quan t i t a t i v e l y determined the products by amino acid analysis, we found that 9 to 11 residues were removed by carboxypeptidase A. The removal was not quantitated i n t h i s study but reaction conditions were such that close to quantitative removal of the COOH-terminus was l i k e l y . This was the case whether the C-TM was fluorescently l a b e l l e d or not. The presence of any endopeptidase a c t i v i t y was checked for by observing migration of fluorescently l a b e l l e d C-TM and NPTM on SDS-PAGE. There were only single bands of fluorescence i n each lane. Subsequent staining with Coomassie Blue also revealed only single bands, again i n d i c a t i n g that the only alterations i n the tropomyosin were at the COOH-terminus. Truncated p l a t e l e t tropomyosin (P-TM*) was produced i n the same way, except only 4 residues were removed (Clark and 39 Burtnick, 1988). This included Cys-246 which l e f t Cys-153 as the s i t e f o r l a b e l l i n g by s u l f h y d r y l - s p e c i f i c probes. Concentrations of unlabelled C-TM and NPTM samples i n solut i o n were determined spectrophotometrically using a molar absorption c o e f f i c i e n t of 22760 M"1 cm"1 at 277nm (McCubbin and Kay, 1969), with the assumption that the absorption c o e f f i c i e n t would remain unchanged as no Tyr residues were removed by the carboxypeptidase A. Other absorption c o e f f i c i e n t s (280nm) used were 0.24 ml mg"1 cm"1 f o r P-TM (C6te and S m i l l i e , 1981b), 1.23 ml mg"1 cm"1 for DNase I (Hitchcock et al., 1976), 0.62 ml mg"1 cm"1 for G-a c t i n (Gordon et al., 1976) and 1.4 ml mg - 1 cm - 1 for g e l s o l i n (Ruiz S i l v a and Burtnick, 1990). 2.1.3. Fluorescent Labelling of Tropomyosins Fluorescently l a b e l l e d C-TM was prepared by i n i t i a l d i a l y s i s i n 150mM KC1, lOmM T r i s , 5mM DTT, pH 8.0, overnight at 4°C. The C-TM was then dialysed for 4 to 5 hours against 150mM KC1, lOmM T r i s , pH 8.0 (no DTT), aft e r which either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) (Molecular Probes) dissolved i n a minimal volume of 2V,N-dimethylformamide, was added to an approximate 10-fold molar excess over C-TM. The reaction was allowed to proceed for 2 to 4 hours at 37°c i n the dark. The reaction mixture was then centrifuged at 15,000 xg for 10 minutes to remove pr e c i p i t a t e d reagent and 40 the supernatant dialysed overnight against 150mM KC1, 20mM Mops, pH 7.0. Residual unreacted reagent was removed by gel f i l t r a t i o n through BioGel P2 (BioRad). La b e l l i n g of C-TM with dansyl chloride (DNS-C1) was car r i e d out i n 150mM KC1, 40mM Na borate, pH 8.0 (Burtnick and Bhangu, 1986), since amine-containing buffers would react with the dansyl chloride. P l a t e l e t tropomyosin (P-TM) was l a b e l l e d i n e s s e n t i a l l y the same way as C-TM except a lower i n i t i a l concentration of DTT was required (2mM) for l a b e l l i n g at Cys-24 6 (Burtnick et a l . , 1986). Extent of l a b e l l i n g was quantified by independent determinations of the amounts of labe l and protein i n a sample solu t i o n . Concentrations of fluorescent labels were determined using molar absorption c o e f f i c i e n t s of 2.2 x 10 4 M"1cm_1 at 344nm for Py (Kouyama and Mihashi, 1981), 1.29 x 10 4 M-icm - 1 at 360nm for AD (Prendergast et al., 1983) and 4.3 x 10 3 M-1cm_1 at 340nm for DNS (Hiratsuka and Uchida, 1977). Protein concentrations of l a b e l l e d samples were determined using the method of Bradford (1976) as adapted for use with the BioRad protein staining reagent. 2.2. PHYSICAL METHODS Steady state fluorescence spectra were c o l l e c t e d using a Perkin-Elmer LS 5B luminescence spectrometer connected to a Perkin-Elmer 7500 computer. Fluorescence p o l a r i z a t i o n 41 values were determined using PTPOL software (Perkin-Elmer) and a p o l a r i z a t i o n accessory for the LS 5B fluorimeter. Temperature control was achieved with a c i r c u l a t i n g water bath (Haake). Absorbance measurements were made with a Perkin-Elmer Lambda 4B spectrophotometer. C i r c u l a r dichroism data were c o l l e c t e d using a modified Jasco J-20 spectropolarimeter (Landis Instruments) thermally regulated with a c i r c u l a t i n g water bath (Grant). 15 minutes was the thermal e q u i l i b r a t i o n time between measurements. C a l i b r a t i o n of the instrument was c a r r i e d out using a 10mm c e l l f i l l e d with D-10-camphor sulphonic acid (0.06% w/v i n H20) which has an e l l i p t i c i t y at 290nm (8290) of + 0.188 degrees (Chen and Yang, 1977). A 1cm d e f l e c t i o n on the chart recorder f o r the standard sample was found to be 3.123 x 10" 4 degrees. One could then measure the e l l i p t i c i t i e s of samples by measuring the d e f l e c t i o n of the pen on the chart recorder. Molar e l l i p t i c i t i e s ([8]), i n units of degrees cm2 dmole - 1/ were calculated using: [6] = 6M/10cd (2.1) where 6 i s observed e l l i p t i c i t y (degrees), c i s concentration (g/cm3), d i s pathlength i n cm and M i s molecular weight (g/mole) of the o p t i c a l l y active component (163 g/mole for a tyrosine residue). The molar e l l i p t i c i t y is- d i r e c t l y r e l a t e d to the difference i n the extinction 42 c o e f f i c i e n t s of the sample for l e f t and right c i r c u l a r l y p o l a r i z e d l i g h t by the expression: [0] = 3,300 (e x -e r) (2.2) V i s c o s i t y measurements were made with a #100 c a p i l l a r y viscometer (Cannon-Manning) at 27.5°C. Times for samples to move between two points i n the viscometer under the force of gravity were measured i n t r i p l i c a t e . The r a t i o of times for sample and solvent i s taken to be the r e l a t i v e v i s c o s i t y ( T j r e l ) , with the solvent having T j r e l = 1. Iodination of tropomyosin was ca r r i e d out i n the laboratory of Dr. Don Brooks following the method of Markwell (1982) as described by Olal (1990). 43 3. RESULTS AND DISCUSSION : ALTERED COOH-TERMINUS OF C-TM 3.1. EFFECT OF CARBOXYPEPTIDASE A In t h i s section of the thesis, the response along the tropomyosin rod to removal of the 11 residues from i t s COOH-terminus by carboxypeptidase A was studied using c i r c u l a r dichroism and s p e c i f i c fluorescent l a b e l l i n g with acrylodan and pyrene. 3.1.1. C i r c u l a r Dichroism of C-TM and NPTM. 3.1.1.a. Peptide Bond CD. The extent t o which removal of the COOH-terminus affected the cooperativity of unfolding of C-TM was investigated by observing CD at 222nm, a measure of the amount of a-helix i n each sample. Thermal melting curves of the sort i n Figure 15 are common i n TM l i t e r a t u r e (Lehrer, 1978; C6te et a l . , 1978; Mak and Sm i l l i e , 1981a). The data are presented as the r a t i o of measured e l l i p t i c i t i e s at temperature, T, to the e l l i p t i c i t y at the s t a r t i n g temperature (10°C). This results i n plots for both C-TM and NPTM s t a r t i n g at a value of 1.0 at 10°C, even though t h e i r respective a-helix contents are actually 96% and 92% (Mak and S m i l l i e , 1981a). The temperature dependence of 8/810 44 1.2 1.0 0.8 2 0.6 CD 0.4 CD 0.2 0.Q a • a • a • D • a • a • 0 10 20 30 40 50 60 70 Temperature (°C) FIGURE 15. a) Change i n normalized e l l i p t i c i t i e s (222nm) of oxidized C-TM (•) and NPTM (•) with temperature. 4uM samples i n 150mM KC1, lOmM T r i s , pH 8.0. Noise on d i g i t a l v o l t meter was i 0.03V for 10V f u l l scale d e f l e c t i o n . 0.5mm pathlength c e l l . 45 0 10 20 30 40 50 60 70 Temperature (°C) F I G U R E 1 5 T b) Change i n normalized e l l i p t i c i t i e s (222nm) of AD-C-TM (•) and AD-NPTM («» with temperature. 4uM samples i n 150mM KC1, lOmM T r i s , pH 8.0. 46 r e f l e c t s the cooperativity of the h e l i x to random c o i l t r a n s i t i o n i n C-TM and NPTM. Figure 15a shows the melting curves of C-TM and NPTM i n 150mM KC1, lOmM T r i s , pH 8.0 and oxidized at Cys-190. Both sets of data exhibit the pr e t r a n s i t i o n (10-35°C) that i s t y p i c a l of oxidized TM at a r e l a t i v e l y high' i o n i c strength (Skolnick and Holtzer, 1986). It can be seen that there i s only the s l i g h t e s t of ef f e c t s oh the cooperativity of unfolding of the a-helix i f the COOH-terminus i s not present. This suggests that the s t r u c t u r a l i n t e g r i t y of the C-TM molecule as a whole i s not seriously disrupted by the loss of the COOH-terminus, confirming the work of Mak and S m i l l i e (1981a). Figure 15b shows the v a r i a t i o n of 6 / 6 1 0 with temperature of AD-C-TM and AD-NPTM. The cooperativity of melting of these l a b e l l e d species i s almost i d e n t i c a l . There i s no p r e t r a n s i t i o n evident because the probes attached at Cys-190 preclude the p o s s i b i l i t y of a d i s u l f i d e bond. Figure 16 represents the e f f e c t of addition of carboxypeptidase A on the 222nm CD signal from C-TM as the enzyme removes residues from the COOH-terminus. Carboxypeptidase A was added to C-TM i n 150mM KC1, lOmM T r i s , pH 8.0, at a r a t i o of 1/50 (w/w) and contributed a n e g l i g i b l e amount to the 222nm CD si g n a l . After approximately 3 hours the signal had decreased 6-8% as the re s u l t of the action of carboxypeptidase A, consistent with the r e s u l t s of Mak and S m i l l i e (1981a). 47 10-CD > CD t r L J J o--10 0 Time (hours) 2.8 FIGURE 16. Change i n e l l i p t i c i t y at 222nm of a sample of C-TM (5UM) with carboxypeptidase A 50/1 (w/w) added at t = 0 (bottom line) i n 150mM KC1, lOmM T r i s , pH 8.0. Top l i n e i s buffer blank. 3.1.1.b. Tyrosyl CD. CD from tyrosine residues along the TM chain should provide information that i s more s p e c i f i c than polypeptide backbone CD measured near 222 nm. At the same time, i t w i l l be more general than fluorescence from a probe at a single l o c a t i o n on the chain. Of the six tyrosines, i n each TM chain, f i v e are found i n the region from Tyr-162 to the COOH-terminus, and four are found at positions beyond Cys-190. 48 Samples of oxidized TM and NPTM i n physiological i o n i c strength solutions were placed i n 5 mm c e l l s and t h e i r e l l i p t i c i t i e s at 280 nm were followed as a function of temperature (Figure 17). The p r o f i l e s c l o s e l y resemble those reported i n thermal denaturation studies of oxidized TM monitored by peptide backbone CD at 222 nm and by tyrosine fluorescence p o l a r i z a t i o n (Lehrer, 1978). There i s a pr e t r a n s i t i o n that i s e s s e n t i a l l y complete by 30°C followed by a main unfolding t r a n s i t i o n centered near 45°C. Up to the end of the pre t r a n s i t i o n , the signal from NPTM f a l l s further, and at a few degrees lower temperature, than does the s i g n a l from TM. The thermal denaturation curves for TM and NPTM converge i n the region of the main- unfolding t r a n s i t i o n . The lower tyrosine e l l i p t i c i t i e s that we observe i n the room temperature region are consistent with the lower cc-helical content, estimated from e l l i p t i c i t i e s at 222 nm, of NPTM r e l a t i v e to TM at those temperatures (Mak and Sm i l l i e , 1981a). At temperatures near to and above 45°C, the thermal denaturation curves determined for TM and NPTM using e l l i p t i c i t i e s at 222 nm are indistinguishable (Mak and Sm i l l i e , 1981a), as i s the case for our tyrosine CD p r o f i l e s . The CD signal at 280 nm i s an average of the responses of the 6 tyrosine residues on each TM chain (Bullard et al., 1976; Nagy, 1977; Holtzer et al., 1989). The tyrosines are found at positions 60, 162, 214, 221, 261, and 267 (Lewis and S m i l l i e , 1980). As f i v e of the tyrosine residues are 49 25 CD .E 20 O I5H E E 10-4N E O U ) CD O 00 CM i i » • i 5-o--5-10 20 T — r 30 4 0 50 Temperature(°C) T — r 60 70 FIGURE 17. Tyrosine CD melting curves. Data were recorded at 280 nm f o r oxidized samples of C-TM (O) and NPTM (a) , at 1.8 and 1.2 mg/ml, respectively, i n 150mM KC1, lOmM Tris-HCl, pH 50 found i n the COOH-terminal h a l f of the molecule/ i t i s l i k e l y that the more pronounced p r e t r a n s i t i o n of NPTM (Figure 17) i s a r e s u l t of a decreased r i g i d i t y of the c o i l e d c o i l structure i n that h a l f of the protein r e l a t i v e to what i s found i n in t a c t TM. These res u l t s suggest a model i n which, although the COOH-terminal h a l f of the c o i l e d c o i l structure for NPTM i s less constrained than for inta c t TM, the NPTM as a whole i s s t i l l s t r u c t u r a l l y very s i m i l a r to C-TM. Fluorescent l a b e l l i n g at Cys-190 could p o t e n t i a l l y allow access to information on conformational changes at that s i t e , s p e c i f i c a l l y , and a probe at that residue (e.g., pyrene) should provide a more d e f i n i t i v e signal than probes (e.g., the t y r o s y l rings i n the case of Figure 17) 24 or more residues away. 3.1.2. Py-C-TM and Py-NPTM. Degrees of l a b e l i n g of TM with pyrene were t y p i c a l l y 0.8 to 1.0 Py per chain. Figure 18 shows the e f f e c t s of temperature on fluorescence i n t e n s i t i e s from pyrene monomers (measured at 383 nm) and pyrene excimers (measured at 490 nm) i n samples of Py-C-TM and Py-NPTM at approximately p h y s i o l o g i c a l i o n i c strength (Figure 18a, 150mM KC1, 20mM Mops, pH 7.0) and at low i o n i c strength (Figure 18b, 20mM Mops, pH 7.0). 51 10 20 30 40 Temperature (°C) 50 60 FIGURE 18. a) Thermal e f f e c t s on fluorescence of monomers (open symbols) and excimers (closed symbols) i n samples of Py-C-TM (squares) and Py-NPTM (triangles). 1 UM samples of Py-C-TM and Py-NPTM were heated and fluorescence spectra were recorded at various temperatures. Monomer and excimer fluorescence i n t e n s i t i e s were determined, respectively, at 383 and 4 90 nm at each temperature. Conditions were 150mM KC1, 20mM Mops, pH 7.0. Degree of l a b e l l i n g =0.8 Py per chain. E x c i t a t i o n was at 280 nm. 52 10 20 30 40 Temperature (°C) 50 FIGURE 18. b) Same species as i n Figure 18a but i n 20mM Mops, pH 7.0. 53 Several points should be noted i n Figure 18. F i r s t l y , pyrene monomer emission decreases r e l a t i v e l y steeply and monotonically f o r Py-C-TM and Py-NPTM regardless of io n i c strength of the solut i o n . Such behaviour i s largely the res u l t of increased rates of d i s s i p a t i o n of energy from excited state chromophores as a resu l t of increased frequency of c o l l i s i o n with solvent molecules or increased v i b r a t i o n a l mode a c t i v i t y . It can be seen that i n the room temperature region, the monomer fluorescence from Py-C-TM i s greater than the monomer fluorescence for Py-NPTM. This i s due to the f l e x i b i l i t y of each structure determining the proportion of pyrene that w i l l be involved i n excimer formation. As a f i r s t approximation, the more excimer fluorescence there i s , the less monomer fluorescence that can be produced i f the number of pyrenes remains unchanged. Secondly, excimer fluorescence l e v e l s change with temperature i n a more complicated way that r e f l e c t s not only the d i r e c t thermal e f f e c t s on the chromophore, but i n d i r e c t e f f e c t s through thermally-induced changes i n the structure of the polypeptide chains to which the chromophores are bound. Most s t r i k i n g l y , excimer fluorescence i n t e n s i t i e s from Py-C-TM i n phys i o l o g i c a l i o n i c strength solutions decrease slowly as the temperature i s raised from 15°C through about 33°C, but then increase markedly to a maximum near 38°C. Then they f a l l again as temperature increases. Similar behavior has been reported recently for rabbit s k e l e t a l TM l a b e l l e d with pyrene ( I s h i i and Lehrer, 1990) 54 and interpreted i n terms of p a r t i a l unfolding of the Cys-190 region near 38°C, which reduces constraints placed by the c o i l e d c o i l structure on the approach of an excited state pyrene at Cys-190 to a neighboring ground state pyrene at Cys-190 on the second TM chain. Complete separation of the two chains at higher temperatures abolishes excimer formation (also see I s h i i and Lehrer, 1985). The r e l a t i v e i n s t a b i l i t y of the Cys-190 region of TM i n solutions of moderate to high i o n i c strength i s well documented (Woods, 1977; Edwards and Sykes, 1980; Williams and Swenson, 1981; Betteridge and Lehrer, 1983; Graceffa and Lehrer, 1984). The absence of a d i s t i n c t maximum i n excimer fluorescence near 38°C i n Py-C-TM at low i o n i c strength i s consistent with the absence of a pr e t r a n s i t i o n i n CD studies of thermal denaturation of TM i n low i o n i c strength solutions (Betteridge and Lehrer, 1983). Thirdly, Py-NPTM produces changes i n excimer fluorescence with temperature that are completely d i f f e r e n t than those for in t a c t Py-C-TM. At low temperatures the excimer emission i s much higher than i n Py-C-TM, in d i c a t i v e , i n terms of the Graceffa and Lehrer (1980) proposal, of a higher proportion of the more open s t r u c t u r a l form of the c o i l e d c o i l near Cys-190 for Py-NPTM than for Py-C-TM. As temperature increases, the fluorescence drops o f f steeply near 25°C, suggestive of chain separation i n that region at a much lower temperature than i n Py-C-TM. The fact that the only difference between the two molecules i s at the COOH-55 terminus suggests that the structure at the COOH-terminus acts to hold the Cys-190 region i n a s p e c i f i c conformation that responds i n a s p e c i f i c way to increases i n temperature. On removal of the COOH-terminus, the Cys-190 region assumes a more open conformation that i s less stable to increases i n temperature than i n in t a c t Py-C-TM. Fourthly, i n low i o n i c strength solutions near room temperature, excimer fluorescence l e v e l s are lower and monomer fluorescence l e v e l s are higher than observed at higher i o n i c strengths. This may be caused by excimer formation being, i n part, e n t r o p i c a l l y driven and, so, being more favorable at high i o n i c strengths. F i n a l l y , fluorescence emission spectra observed for Py-C-TM and Py-NPTM i n the higher i o n i c strength buffers, which provided data for Figure 18a, could be changed completely and r e v e r s i b l y to spectra c h a r a c t e r i s t i c of those l a b e l l e d species i n low i o n i c strength buffers by d i a l y s i s . A more concise way of representing the data i s as excimer/monomer r a t i o vs. temperature (Figure 19), which retains the e s s e n t i a l features of Figure 18 while reducing the number of curves by 50%. When the same experiment was performed on a sample of Py-NPTM that had not been dephosphorylated p r i o r to treatment with carboxypeptidase A, there was a small but s i g n i f i c a n t peak i n the excimer/monomer r a t i o near 38°C (Figure 19b). This peak was reduced to a small shoulder i n the sample that had been dephosphorylated (Figure 19a, data from Figure 18a). This o p 1.2 1.0 -0.8 56 o z o 0.6 O X Ul 0.4 o o o O O O ° A A A 0.2 0.0 10 20 30 40 TEMP (t) 50 60 O F < O z o u X U) FIGURE 19. a) Data from Figure 18a presented as a r a t i o of excimer to monomer fluorescence for Py-C-TM ,(0) and Py-NPTM (A) . 13) 1.4 1.2 -1.0 0.8 0.6 0.4 0.2 0.0 o o O - A A ° A * A * oB 10 20 30 40 TEMP (*C) 50 60 FIGURE 19, b) Py-C-TM (O) and Py-NPTM (A) samples that were not treated with alkaline phosphatase p r i o r to l a b e l l i n g with Py. Conditions as for Figure 18a. Degree of l a b e l l i n g = 0.9 Py per chain. 57 shoulder may be due to the presence of some undigested Py-C-TM, the r e s u l t of the i n a b i l i t y of carboxypeptidase A to remove residues beyond and including phosphorylated Ser-283, which occurs normally i n TM preparations at l e v e l s of approximately 20% (Mak et al., 1978). Therefore, a l l other data presented i n t h i s chapter were obtained with TM that had been dephosphorylated (Heeley et al., 1987). An a l t e r n a t i v e representation of the difference i n fluorescence properties of Py-C-TM and Py-NPTM at 37°C i n solutions of p h y s i o l o g i c a l i o n i c strength i s provided i n Figure 20. The emission spectrum of a sample of Py-C-TM was recorded (Figure 20a) and then an aliquot of carboxypeptidase A was added. The change i n fluorescence at 490 nm ( F 4 9 0 ) was monitored with time (Figure 20b) . After 3 hours the spectrum was recorded again (Figure 20a). The data i l l u s t r a t e the change i n conformation at Cys-190 as carboxypeptidase A removes amino acid residues from the COOH-terminus. A control experiment i n which buffer that contained no carboxypeptidase A was added to Py-C-TM confirmed that photobleaching was not a factor i n the r e s u l t s . The "before" and " a f t e r " spectra were i d e n t i c a l to each other f o r the control. The absence of photobleaching i s a t t r i b u t a b l e to the low i n t e n s i t y of the ex c i t a t i o n source (a pulsed 8.3 W Xe discharge lamp) i n the system. 58 L d O -z. L d o CO L d CH O 3 L d > f— < - J L d CH l O O n 360 400 440 480 WAVELENGTH (nm) 520 F I G U R E 20. a) Fluorescence spectra of a 1 UJM Py-C-TM sample i n 150mM KC1, 20mM Mops, pH 7.0, recorded before ( ) and a f t e r ( ) 3 hours of digestion with carboxypeptidase A at a 1/50 (w/w) r a t i o to TM. Temperature = 37°C. Degree of l a b e l l i n g = 0.8 Py per chain. E x c i t a t i o n was at 280 nm. | IOO-I »» Ld O CO Ld ^ % E O c ZD CD L d > L d o-o —r-10 20 30 40 TIME (min) y/i 1 170 180 F I G U R E 20. b) Decrease of excimer fluorescence on digestion of sample from a) with carboxypeptidase A added at t = 0. \ 59 300 400 500 T /77 (K/cpo i se) 700 Figure 21. Perrin p l o t s for 2 UM samples of AD-C-TM (•) and AD-NPTM (•) i n 150mM KC1, 20mM Mops, pH 7.0. Pol a r i z a t i o n values of AD were measured at various temperatures. Degree of l a b e l l i n g = 1.0 AD per chain. E x c i t a t i o n was at 360 nm. Emission was detected at 510 nm. 3.1.3. AD-C-TM and AD-NPTM. Figure 21 shows Perrin plots for AD-C-TM and AD-NPTM i n solutions near physiological i o n i c strength, constructed 60 by measuring acrylodan p o l a r i z a t i o n at d i f f e r e n t temperatures. The AD-TM data agree well with those of Lehrer and I s h i i (1988), with extrapolation to T/T| = 0 giving l i m i t i n g p o l a r i z a t i o n values, p Q, of 0.40 i n both cases. If the p Q i s approximated using AD-C-TM i n 95% glycerol at 10°C, i t i s found to be 0.39. The fact that these two values are e s s e n t i a l l y the same suggests that there i s n e g l i g i b l e contribution from probe motion, such as rapid rotation about the single covalent bond that attaches AD to TM, to depolarization of the acrylodan fluorescence. In other words, the acrylodan i s held firmly by the tropomyosin molecule. Assuming a fluorescence l i f e t i m e of 1.9ns (Lehrer and I s h i i , 1988), use of the Perrin equation gives a ro t a t i o n a l c o r r e l a t i o n time for the protein of 6.4ns. The end-over-end tumbling of tropomyosin occurs on the us timescale (Hvidt et al., 1983) which suggests that the acrylodan i s probing the rotation of the TM around the axis that extends the length of the molecule. The lower p o l a r i z a t i o n values (higher 1/p values i n Figure 21) for acrylodan attached to Cys-190 of NPTM r e l a t i v e to in t a c t C-TM at a l l temperatures studied shows acrylodan to be less constrained on AD-NPTM than on AD-C-TM. This i s consistent with the Py res u l t s i n suggesting that the COOH-terminus i n some way restrains the Cys-190 region. Removal of the COOH-terminus with carboxypeptidase A relaxes these r e s t r a i n t s . Similar Perrin plots were obtained for AD-l a b e l l e d samples i n low i o n i c strength solutions (results not shown). Unlike the Py-C-TM case i n moderate concentration s a l t solution, AD-TM p o l a r i z a t i o n behaviour with temperature does not show a dramatic anomaly near 38°C. This probably r e f l e c t s differences i n the b a r r i e r s to excimer formation f o r pyrenes (one excited) on adjacent TM chains, r e l a t i v e to those f o r independent rotation of acrylodan molecules at t h e i r respective Cys-190 s i t e s of attachment. As with Py-C-TM, the e f f e c t s of removal of the COOH-terminus of AD-TM at 37°C i n a solution at p h y s i o l o g i c a l i o n i c strength can be followed d i r e c t l y by measurement of p o l a r i z a t i o n at d i f f e r e n t time i n t e r v a l s (Figure 22). The O.Oh -0.03 i 1 1 1 1 1 1 1 1 0 20 40 60 80 100 120 140 160 180 Time (min) F I G U R E 22. Decrease of AD-C-TM fluorescence p o l a r i z a t i o n on digestion with carboxypeptidase A. P o l a r i z a t i o n from 2\1M AD-C-TM was measured at various times af t e r the addition of carboxypeptidase A to the sample i n a 1/50 (w/w) r a t i o to TM and subtracted from p o l a r i z a t i o n values recorded for a s i m i l a r sample to which only buffer had been added. Conditions were as i n Figure 21. Temperature = 37°C. Data are presented as mean"*^ standard deviation of three p o l a r i z a t i o n measurements at a single temperature. 62 small but s i g n i f i c a n t drop i n p o l a r i z a t i o n again indicates a greater degree of f l e x i b i l i t y of the probe at Cys-190 i n the absence of the COOH-terminus. 3.2 C-TM/NPTM HYBRIDIZATION The nature of the int e r a c t i o n between the COOH-terminus of one C-TM molecule and the NH 2-terminus of an adjacent C-TM that i s responsible for polymerization i s not well understood. The low resolution c r y s t a l structure (15A) suggests a globular domain ( P h i l l i p s et al., 1986), but may not accurately represent the s i t u a t i o n that occurs i n so l u t i o n . As has already been discussed, removal of both COOH-terminal chains of C-TM with carboxypeptidase A completely removes the a b i l i t y to polymerize and bind to a c t i n (Mak and S m i l l i e , 1981a). In t h i s section, hybridization methods discussed i n Chapter 1 (1.4.2.) are used to produce a C-TM species with only one i n t a c t COOH-terminal chain (C-TM*). Using v i s c o s i t y and a c t i n binding assays, the a b i l i t y of t h i s novel species to function as C-TM was investigated. C-TM and NPTM stock solutions were prepared. Test and control samples were equimolar mixtures of C-TM and NPTM i n the presence and absence of lOmM d i t h i o t h r e i t o l (DTT), respectively, with a t o t a l [TM] = 1.0 x 10"5 M (Table I, column 1; Buffer: 20mM Mops) or 2.4 x IO - 5 M (Table I, column 2; Buffer: lOmM Mops). The samples were then heated 63 to 60°C f o r 15 minutes and cooled by eithe r leaving at room temperature or quenching i n an ice/water bath (Holtzer et a l . / 1984). V i s c o s i t y measurements were then c a r r i e d out at 27.5°C. The res u l t s obtained are presented i n Table I. The presence of DTT i n the tes t samples allows complete separation of the in d i v i d u a l C-TM and NPTM chains at 60°C/ which i s not possible i n the controls due to the d i s u l f i d e bonds. In the controls t h i s forces renaturation to the i n i t i a l state upon cooling/ while allowing the p o s s i b i l i t y of hybrid formation i n the tes t samples. As can be seen from Table 1/ the tes t solutions a f t e r treatment display a decreased v i s c o s i t y r e l a t i v e to the controls. We interpret t h i s as r e f l e c t i n g formation of NPTM/C-TM hybrids i n the t e s t cases and the hybrids being unable to polymerize. TABLE I SAMPLE T [ r c l ( + / - 0 . 0 1 ) Buffer 1.00 1.00 NPTM 1.09 1.13 C-TM 1.17 2.30 Control (Quenched) 1.15 1.36 Control (Slow cooling) 1.15 1.42 Test (Quenched) 1.09 1.29 Test (Slow cooling) 1.10 1.35 64 We s t a r t by making the assumption that, upon cooling, the single chains of C-TM and NPTM i n the hybrid mixture are s u f f i c i e n t l y a l i k e to be indistinguishable, so that there exists an equal p r o b a b i l i t y of recombination between l i k e or unlike partners. In other words, the r a t i o of species w i l l change thus: C-TM + NPTM > C-TM + C-TM* + NPTM 50% 50% 25% 50% 25% This assumption i s supported by the experimental evidence that the NH 2-terminal half of C-TM i s much more stable than the COOH-terminal half (Woods, 1977; Pato et al., 1981) and that as few as 30 residues very near the NH2-terminus are involved i n the i n i t i a l i n t e r a c t i o n as the chains are cooled (Brown and Schachat, 1985). Therefore, since the chains of C-TM and NPTM are i d e n t i c a l except for removal of the COOH-terminus, a given molecule w i l l be considerably renatured with what i t w i l l perceive to be a perfect match before the COOH-termini come together. In the control samples, 50% of the c o i l e d c o i l species present should be polymerizable (the C-TM molecules). In the te s t mixtures, 50% of the species should be C-TM* (hybrids). Therefore, i f C-TM* i s f u l l y able to polymerize, then 75% of the molecules present should be polymerization-competent and v i s c o s i t i e s of the hybrid sample should have exceeded those of the control samples. From Table I i t can be seen that t h i s i s not the case and i t can be concluded that a C-TM molecule requires that the COOH-termini of both of i t s 65 chains be i n t a c t i n order for the end-to-end int e r a c t i o n necessary f o r polymerization to occur normally. Although the C-TM* species i s expected to be thermodynamically le s s stable than C-TM due to the unpaired 9 to 11 COOH-terminal residues of the intact chain on C-TM*, i t may be trapped k i n e t i c a l l y as a re s u l t of the proposed f o l d i n g pathway. However, a complication i s that Lehrer and Qian (1990) found that although gizzard TM homodimers (oca and BB) are stable to chain exchange below 25°C, at 37-40°C the native aP heterodimer w i l l form spontaneously v i a an unspecified chain exchange mechanism. Therefore, chain exchange to reach thermodynamic equilibrium may be possible. When our 10J1M C-TM/NPTM tes t mixture was incubated for 15 minutes at 37°C then cooled, there was no difference i n v i s c o s i t y from the hybrid samples that had been cooled d i r e c t l y from 60°C. This suggests that no chain exchange had taken place, or the v i s c o s i t y would have approached that of the control samples. To check whether the hybridization produced species that are i n reg i s t e r , the procedure of Lehrer (1975) using 5,5'-dithiobis(2-nitrobenzoate) [(NbS) 2] was employed. After the hybrid and control mixtures were removed from the viscometer, they were dialysed overnight against 20mM Mops, pH 7.0, to remove the DTT present. I f the two chains i n the c o i l e d c o i l were i n regi s t e r , t h i s treatment would r e s u l t i n d i s u l f i d e bond formation at adjacent Cys-190 residues (Lehrer, 1978). I f there were any free -SH groups produced 66 i n the hybridization process, as a re s u l t of the chains being out of re g i s t e r , they would react with (NbS) 2 and release 2-nitro-5-thiobenzoate (NbS), which absorbs at 412nm. It was found that there was no change i n A 4 1 2 i n the control or hybrid mixtures, from which we conclude that a l l renatured species were i n r e g i s t e r . Attempts to carry out actin-binding assays using 1 2 5 I - l a b e l l e d NPTM were not productive because of the presence of two d i f f e r e n t l a b e l l e d species, 1 2 5I-NPTM and 125J_C-TM*, i n the hybrid mixture, as well as the presence of C-TM, which p o t e n t i a l l y could increase binding of NPTM and C-TM* to a c t i n . It i s clear that actin-binding studies would be best c a r r i e d out on C-TM* p u r i f i e d from the hybrid mixture. The r e s u l t s i n t h i s section suggest that both COOH-termini of a C-TM molecule are required for function and that a form of C-TM with only one COOH-terminus intact i s as incapable of function as NPTM. Whatever the nature of the int e r a c t i o n between the four chain ends i n the overlap region of adjacent C-TM molecules (two COOHrtermini and two NH 2-termini), the removal of one of the COOH-termini has a much greater e f f e c t on that i n t e r a c t i o n than might be expected. Cho et al. (1990) produced a mutant TM with residues 1-9 of the NH 2-terminus deleted and found that, even i n the presence of troponin, i t was unable to bind to F-actin, unlike NPTM (Heeley et a l . , 1987). It would be in t e r e s t i n g to see i f hybridizing t h i s mutant with C-TM was 67 possible and i f a species with only one inta c t NH 2-terminus was any more polymerizable than C-TM* and i f i t could be induced to bind to F-actin i n the absence or presence of troponin. 3 . 3 . C-TM/P-TM HYBRIDIZATION Taking the hybridization methodology one step further, the p o s s i b i l i t y of hybridizing C-TM and P-TM can be considered. The information obtainable from t h i s system does not address questions about TM function but does pertain to the genetics that leads to d i v e r s i t y i n TMs. The main s i m i l a r i t i e s between these TMs are t h e i r c o i l e d c o i l structure and the large section of sequence that i s conserved i n the two proteins (Figure 9a). The main differences are the length (P-TM chains are 37 residues shorter than C-TM) and the non-conservative substitutions at the NH2- and COOH-termini. These are the factors that have to be balanced and ultimately are what decide the outcome of the hybridization study. The only experiment that suggested that the P-TM/C-TM species could be made involved the use of P-TM l a b e l l e d with pyrene. P-TM i n 150mM KC1, lOmM T r i s , pH 8.0, was reduced overnight with 5mM DTT that was subsequently removed by d i a l y s i s . 8M Guanidine hydrochloride (GuHCl) then was added to a f i n a l concentration of 4M to separate the P-TM chains. N-(1-pyrenyl)iodoacetamide (PIA) i n DMF was added i n excess 68 and reacted with the P-TM for 2 hours. The Py-P-TM was then dialysed exhaustively against 600mM KC1, 50mM Mops, pH 7.0. Hybridization with C-TM was c a r r i e d out, as i n the previous section, by heating to 60°C i n the presence of 10mM DTT, with the control samples being mixed only a f t e r quenching to 4°C. Hybridization was detected by observing the loss of excimer fluorescence of the t e s t samples when compared to the controls. Excimer emission could only come from c o i l e d c o i l s formed between two Py-labelled P-TM chains, where the Cys-153 and Cys-246 positions on adjacent chains would be i n r e g i s t e r . Py-P-TM also was hybridized with P-TM as control to f i n d the maximal expected reduction i n excimer. The data contained i n Figures 23 and 24 show the outcome of the experiment and are summarized i n Table I I . Figure 23 contains the pyrene emission spectra from equimolar mixtures of Py-P-TM and P-TM or C-TM. It can be seen that the Py-P-TM hybridizes s i g n i f i c a n t l y with unlabelled P-TM giving a reduction i n F 4 9 0/F 333 from 0.61 to 0.40, while the attempt to hybridize with C-TM produced a reduction i n F 4 9 0/F3 83 from 0.63 to 0.59. I f the assumption i s made that the renaturation of Py-P-TM and P-TM i s random, using the same reasoning as f o r C-TM and NPTM, and that every Py-P-TM has one and only one Py attached per chain, then t h i s r e s u l t suggests that a maximum of 10% of the Py-P-TM/C-TM mixture a f t e r hybridization i s hybrid molecules. 10% w i l l be an overestimate because our P-TM was not f u l l y l a b e l l e d . Figure 24 shows what happens when the amount of 69 TABLE I I SAMPLE 383-(1:1) (1:5) Py-P-TM/P-TM (Control) 0.61 0.64 Py-P-TM/P-TM (Test) 0.40 0.23 Py-P-TM/C-TM (Control) 0.63 0.65 Py-P-TM/C-TM (Test) 0.59 0.46 unlabelled species i s increased to f i v e times that of Py-P-TM. Figure 24a shows that increasing the concentration of P-TM decreases the p o s s i b i l i t y of two Py-P-TM coming together upon renaturation and the l e v e l of excimer drops s i g n i f i c a n t l y . Figure 24b also shows a marked decrease i n the excimer fluorescence when the concentration of C-TM i s increased/ which confirms that i t i s possible to produce the Py-P-TM/C-TM species. The fact that rapid cooling, as opposed to slow cooling, was required to produce the hybrid species here suggests i t to be a kin e t i c a l l y - t r a p p e d product. Since the renaturation of two a l i k e TM chains involves the NH 2-termini regions recognizing each other (Brown and Schachat, 1985)/ the difference i n t h i s region between C-TM and P-TM becomes the c r i t i c a l factor i n determining the outcome of the hybridization reaction. Lehrer and Qian 70 I60i o LxJ o CO LU Ctl o 3 0 a) U J > U J or 160-1 b) 0 360 4 0 0 4 4 0 480 Wavelength (nm) 520 FIGURE 23. Pyrene emission spectra from 1UM Py-P-TM i n 1:1 molar r a t i o with a) P-TM and b) C-TM i n 600mM KC1, 50mM Mops, lOmM DTT, pH 7.0. Hybrid ( s o l i d lines) and control (dashed lines) samples d i f f e r e d i n that the proteins were mixed before and af t e r the heating/cooling step, respectively. Degree of l a b e l l i n g = 1.1 Py per chain. 71 I60i Ld O -z. o OO U J CY. O 3 0 >I60 cr. b) o 360 400 440 480 520 Wavelength (nm) FIGURE 24. Pyrene emission spectra from 1|1M Py-P-TM i n 1:5 molar r a t i o with a) P-TM and b) C-TM i n 600mM KC1, 50mM< Mops, lOmM DTT, pH 7.0. Hybrid ( s o l i d lines) and control (dashed lines) samples d i f f e r e d i n that the proteins were mixed before and af t e r the heating/cooling step, r e s p e c t i v e l y . 72 (1990) found that rapid cooling of an equimolar mixture of gizzard a and P TMs produced pp-TM and aa-TM, although e q u i l i b r a t i o n at 37°C produced the aP~TM species. The pp-TM formed f i r s t p r e f e r e n t i a l l y , presumably due to preferred i n t e r a c t i o n of the NH 2-termini. This required that the remaining a chains renature with themselves, although the most thermodynamically stable molecule i s aP~TM. In the case of Py-P-TM and C-TM, i t i s the differences i n the NH2-terminal regions that cause P-TM's reduced s e l f -p o l y m e r i z a b i l i t y r e l a t i v e to C-TM (Lewis et al., 1983; Burtnick et al., 1986) and the reason the native species w i l l be the thermodynamic product of hybridization. Only by rapid thermal quenching can the Py-P-TM/C-TM species form and even then only i n low concentration. 73 4. RESULTS AND DISCUSSION : ACTIN-BINDING PROTEINS AND TROPOMYOSIN The i n t e r a c t i o n of actin-binding proteins with tropomyosin i s of in t e r e s t because some a c t i n filaments i n non-muscle c e l l s have tropomyosin associated with them and the actin-TM i n t e r a c t i o n may be modulated by actin-binding proteins (Payne et al., 1986). Also, tropomyosin may modulate the actin-binding protein's i n t e r a c t i o n with a c t i n (C6te, 1983; Payne and Rudnick, 1984)). Another reason for looking at such binary systems i s to observe changes i n the tropomyosin molecule i n order to understand how i t s s t r u c t u r a l s u b t l e t i e s r e l a t e to i t s role i n the regulation of contraction. 4.1. INTERACTION OF DEOXRIBONUCLEASE I WITH TROPOMYOSIN Bovine pancreatic deoxyribonuclease I (DNase I) i s an endonuclease/ the a c t i v i t y of which can be i n h i b i t e d e n t i r e l y by complex formation with monomeric a c t i n (Lazarides and Lindberg, 1974). DNase I also has been reported to form a 2:1 complex with muscle tropomyosin and to i n t e r f e r e with the a b i l i t y of tropomyosin to polymerize i n low i o n i c strength solutions (Payne et al., 1986). This chapter presents the re s u l t s of fluorescence and c i r c u l a r dichroism studies on the in t e r a c t i o n of DNase I with tropomyosin. 74 4.1.1. Fluorescence Studies Three acrylodan-labelled tropomyosin species, AD-C-TM, AD-P-TM, and AD-P-TM* were used to investigate the TM-DNase I i n t e r a c t i o n , with the AD bound at Cys-190, Cys-246, and Cys-153, respectively. Cys-153 of P-TM i s analogous to Cys-190 of C-TM and was l a b e l l e d s p e c i f i c a l l y by p r i o r removal of Cys-246 with carboxypeptidase A (Burtnick et a l . , 1986; Clark and Burtnick, 1988) . Figure 25 shows the AD emission spectra of AD-C-TM i n the absence and presence of DNase I (mole r a t i o 1:2). The e f f e c t of the DNase I on the AD emission p r o f i l e was to cause a lOnm re d - s h i f t with an associated drop i n i n t e n s i t y of about 15%. This e f f e c t was observed for a l l three samples 4 9 0 5 0 0 510 5 2 0 5 3 0 5 4 0 5 5 0 Wavelength (nm) FIGURE 25. Emission spectra of acrylodan-labelled C-TM (lu\M) i n the absence ( ) and presence ( ) of DNase I (2uM) . Samples were dissolved i n 150mM KC1, 20mM Mops, 2mM CaCl 2, O.lmM PMSF, pH 7.0. Degree of l a b e l l i n g = 0.4 AD per chain. E x c i t a t i o n was at 360nm. (Table I I I . ) , despite the fact that the s i t e of AD attachment varied from the COOH-terminus (AD-P-TM) to the ce n t r a l region (AD-C-TM, AD-P-TM*). The data can be interpreted as being due to an increased exposure of the probe to solvent i n the presence of DNase I. This conclusion was supported by quenching AD fluorescence with KI (Figure 26). The slopes of the Stern-Volmer p l o t s increased for a l l three AD-TM samples i n the presence of a two-fold molar excess of DNase I, indi c a t i n g the AD i s more exposed to solvent and, hence, more accessible to iodide ions. Bimolecular rate constants (kCT) f o r quenching have been calculated (Table III) assuming TABLE I I I . Sample P P* nm M " 1 M ~ l • s" - I x 10"8 AD-dithiothrcilol 525 2.79 — 0.068 — AD-P-TM 515 1.29 6.79 0.300 0.167 DNase I/AD-P-TM 525 1.67 8.79 0.203 — A D - C - T M 513 1.97 10.4 0.350 0.181 DNase I/AD-C-TM 522 2.52 13.3 0.269 — A D - P - T M * 511 1.54 8.11 0.320 0.186 DNase I/AD-P-TM* 522 1.77 9.32 0.206 — ([DNase I]/[TM] = 2/1; p* = p o l a r i z a t i o n i n presence of 6M Guanidine Hydrochloride). 76 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 [KQ (M) FIGURE 26. Stern-Volmer plots for iodide quenching of acrylodan-labelled tropomyosins (ljlM) i n the absence (4» and presence O of DNase I (2u\M) i n 150mM KC1, 20mM Mops, 2mM CaCl 2, O.lmM PMSF, pH 7.0. a) AD-P-TM. b) AD-P-TM*. c) AD-C-TM. E x c i t a t i o n was at 360nm and emission at 515nm. 77 a fluorescence l i f e t i m e of 1.9 ns for AD bound to tropomyosin (Lehrer and I s h i i , 1988). The values obtained suggest that the probe on tropomyosin i s somewhat more exposed to the solvent than i t i s on AD-actin (Marriott et al., 1988), consistent with the shorter wavelength emission maximum (492nm) reported f o r AD-actin. Once again, the ef f e c t of DNase I binding was independent of the s i t e of l a b e l l i n g , i n d i c a t i v e of a loosening of the structure at both the COOH-terminus and in t e r n a l s i t e s . Fluorescence p o l a r i z a t i o n measurements (Figure 27) indicate that the binding of DNase I to the AD-TM species r e s u l t s i n a s i g n i f i c a n t drop i n p o l a r i z a t i o n of 25-35%. This magnitude of p o l a r i z a t i o n drop suggests substantial loss of tropomyosin s t r u c t u r a l r i g i d i t y i n the region of the acrylodan probe, as observed using other probes (Burtnick and Racic, 1988; Burtnick and Wong, 1989). Maximal e f f e c t i s reached at a molar r a t i o of DNase I to AD-TM of about 2, which i s the stoichiometry of the pr e c i p i t a b l e complex studied by Payne et al. (1986). This was also the stoichiometry found by Burtnick and Wong (1989) using C-TM l a b e l l e d with 5-iodoacetamidofluorescein. Whether the two DNase I molecules are bound at d i f f e r e n t s i t e s on the TM c o i l e d c o i l or at the same s i t e on the adjacent chains cannot be discerned from t h i s data. The binding i n t e r a c t i o n was confirmed i n another way using 5-(dimethylamino)naphthalene-l-sulfonyl chloride 78 ra c_ ro C D 0.40-0.35 -j : 0.30: • 0.25; 0.20-j 0.15 «• 0.10 0.35-3 0.30 0.25-j 0.20 0.15-3 0.10 035 0.30 0.25 020-| 0.15 -i 0.10 a) • • b) -> 1 1 r T »" • • • a a a • r" c) • • • • • • • • a 0 1 2 3 4 5 [DNase I]/[Tropomyosin] FIGURE 27. Fluorescence p o l a r i z a t i o n changes i n acrylodan-l a b e l l e d tropomyosin (1UM) upon the addition of DNase I O) i n 150mM KC1, 20mM Mops, 2mM CaCl 2/ O.lmM PMSF, pH 7.0. Control measurements were made with the addition of buffer <•). a) AD-P-TM*. b) AD-P-TM. c) AD-C-TM. Each p o l a r i z a t i o n value was the average of 6 measurements. Typical standard deviations were +/- 0.001 i n a) and b) and +/- 0.020 i n c ) . Ex c i t a t i o n was at 360nm and emission at 515nm. 79 (dansyl chloride, DNS) bound to C-TM (Burtnick and Bhangu, 1986). DNS binds covalently to exposed lysi n e groups along the C-TM backbone and up to 16 DNS per TM molecule can be attached. Again, quenching with KI was used, but t h i s time quenching decreased i n the presence of DNase I (Figure 28). This means that the DNS groups on the C-TM are becoming less accessible to the solvent with DNase I present, suggesting that the DNase I p h y s i c a l l y protects some of the DNS molecules on the surface of C-TM. Since the s i t e s of attachment of the DNS molecules are not known, the binding s i t e ( s ) of the DNase I cannot be predicted. DNase I i s a globular protein 257 residues long, with three tryptophan (Trp) residues at positions 155, 179 and 191 that are a l l on or near the surface at one side of the molecule (Oefner and Suck, 1986). Tryptophan fluoresces and since there are no tryptophans i n tropomyosin, the e f f e c t on the i n t e r a c t i o n of tropomyosin with DNase I on the quenching of tryptophan fluorescence with iodide ions could be studied (Figure 29). It was found that the binding of DNase I to C-TM or P-TM reduces the a c c e s s i b i l i t y of these residues to quencher. The Trp residues on DNase I are protected to the same degree upon binding to C-TM or P-TM suggesting that the binding s i t e on DNase I i s the same f o r both TM species. Further examination of the report on the c r y s t a l structure of DNase I, which i d e n t i f i e s the DNA and actin-binding s i t e s (Oefner and Suck, 1986), suggests that the face of the molecule containing the tryptophan i s not part of either the 80 DNA or the actin-binding s i t e . Payne and Rudnick (1986) also suggested that the tropomyosin and actin have d i f f e r e n t binding s i t e s on DNase I because tropomyosin does not affect the endonuclease a c t i v i t y of DNase I while actin does. 0 .0 0 .1 0 .2 0 .3 [KI] (M) FIGURE 28. Stern-Volmer plots for iodide quenching of dansyl-labelled C-TM (lUJM) i n the absence (4» and presence O of DNase I (2uiM) i n 150mM KC1, 20mM Mops, 2mM CaCl 2, O.lmM PMSF, pH 7.0. Degree of l a b e l l i n g = 6.2 DNS per chain. E x c i t a t i o n was at 360nm and emission at 510nm. 81 2.0 F / F o 0.0 0.1 0.2 0.3 [ K I ] ( M ) FIGURE 29. Stern-Volmer p l o t s f o r iodide quenching of tryptophan fluorescence from DNase I (luM) in.the absence (•) and presence of eithe r C-TM (2uM) (•) or P-TM (2uM) (•) i n 150mM KC1, 20mM Mops, 2mM CaCl 2/ O.lmM PMSF, pH 7.0. 4.1.2. C i r c u l a r Dichroism The AD data suggests that there i s a s i g n i f i c a n t l o c a l conformational change i n the TM species upon binding of DNase I, with the p o l a r i z a t i o n values dropping almost to le v e l s for the denatured protein (Table I I I ) . This was investigated further using CD, which can detect changes i n 82 the a-helix content of TM from the changes i n e l l i p t i c i t y at 220nm. Since CD i s an absorption phenomenon and there i s a l i n e a r r e l a t i o n s h i p between absorbance and concentration, CD signals from two sources can be added to give the t h e o r e t i c a l signal of a mixture of the two species, assuming no i n t e r a c t i o n . Figure 30a shows the i n d i v i d u a l spectra of C-TM and DNase I which, when added, give the dashed l i n e i n Figure 30b. However, as can be seen from the experimental r e s u l t ( s o l i d l i n e , Figure 30b), the e l l i p t i c i t y of the 2:1 DNase I/C-TM mixture i s s i g n i f i c a n t l y less than i f there had been no change i n a-helix content upon binding of DNase I to C-TM. The change for AD-labelled or unlabelled C-TM was, on average, an 18% decrease from the expected value assuming no int e r a c t i o n , and f o r P-TM (labelled or unlabelled) i t was an average of 14% l e s s . A difference between observed and calculated e l l i p t i c i t i e s indicates a conformational change has occurred i n one or both of the proteins as a re s u l t of the i n t e r a c t i o n (e.g., Burtnick and Kay, 1976). As about 80% of the t o t a l e l l i p t i c i t y (calculated) of the mixture at 220nm would have been contributed by the TM, i t i s probable that most of the decrease i n the e l l i p t i c i t y i s due to a reduction of the a-helical content of the TM as a r e s u l t of DNase I binding. The s p e c i f i c region(s) on TM that have l o s t a - h e l i c a l structure cannot be ascertained from the CD data alone but, invoking the fluorescence data, the Cys-190 83 Wavelength (nm] FIGURE 30. C i r c u l a r dichroism spectra of proteins i n 150mM KC1, 20mM Mops, 2mM CaCl 2, O.lmM PMSF, pH 7.0. A) DNase I (dotted line) and cardiac tropomyosin ( s o l i d line) at 2.0|iM and 1\UA, respectively. B) Observed ( s o l i d line) and predicted (dashed line) spectra f o r a solution containing 2. OHM DNase I and 1|1M C-TM. 84 region of C-TM and the Cys-153 and Cys-246 regions of P-TM may be s i t e s of a considerable proportion of the structure l o s s . It i s i n t e r e s t i n g to note that a s i m i l a r study on the DNase I/actin complex (Ajtai and Venyanimov, 1983) showed e s s e n t i a l l y no net structure change upon complex formation. It has been known f o r some time that DNase I i s i n h i b i t e d by G-actin (Lazarides and Lindber.g, 1974) by formation of a 1:1 complex, but the phys i o l o g i c a l purpose of t h i s i n t e r a c t i o n remains a mystery. DNase I i s also able to depolymerize F-actin (Hitchcock et a l . , 1976) and t h i s i s p a r t i a l l y i n h i b i t e d when tropomyosin and troponin are bound to the a c t i n . The a b i l i t y of DNase I to i n t e r f e r e with the end-to-end overlap of tropomyosin may be required to s t r i p i t from the F-actin which can then be depolymerized at the filament end by other DNase I molecules. The fact that the Trp quenching studies support the idea of d i f f e r e n t binding s i t e s on DNase I for act i n and TM suggests that a ternary complex may be an intermediate i n t h i s process. The observation that DNase I aff e c t s both tropomyosin and a c t i n suggests that other known actin-binding proteins may also regulate tropomyosin as part of t h e i r function. 4.2. INTERACTION OF GELSOLIN WITH TROPOMYOSIN Ge l s o l i n i s an act i n binding protein that i s d i s t r i b u t e d i n a wide variety of animal c e l l s including cultured c e l l s (Pollard and Cooper, 1986). The protein shows 85 three functions - severing of a c t i n filaments, nucleation of a c t i n polymerization, and capping of the barbed end of a c t i n filament. The actin-severing a c t i v i t y i s regulated i n a calcium-dependent way. Studies on the i n t e r a c t i o n between g e l s o l i n and a c t i n i n the presence of muscle and p i g p l a t e l e t tropomyosins have shown that the severing a c t i v i t y of the g e l s o l i n i s retarded but not blocked (Pruliere et a l . / 1986a). Different tropomyosin isoforms from cultured rat c e l l s show d i f f e r e n t degrees of i n h i b i t i o n of a c t i n -severing by g e l s o l i n (Ishikawa et a l . , 1989) ranging from p a r t i a l i n h i b i t i o n by high molecular weight' TMs to no i n h i b i t i o n by low molecular weight TMs. The g e l s o l i n used i n these experiments was p u r i f i e d from horse plasma by Scott Reid i n our laboratory. It i s s i m i l a r i n chemical composition and physical properties to other mammalian plasma g e l s o l i n s , having a molecular weight of nearly 85,000 g/mole (Ruiz S i l v a and Burtnick, 1990). The gelsolin/tropomyosin i n t e r a c t i o n was studied to obtain further information on the function of tropomyosin i n regulating a c t i n filament length and to compare the i n t e r a c t i o n to that of tropomyosin with DNase I. 4.2.1. Viscosity As with the DNase I/C-TM interaction, the i n t e r a c t i o n of g e l s o l i n with C-TM reduces the low i o n i c strength p o l y m e r i z a b i l i t y of the C-TM but only i f ImM C a 2 + i s present 86 (Figure 31). It can be seen that only when the TM/gelsolin r a t i o i s less than 10:1 does the r e l a t i v e v i s c o s i t y Cn re l) begin to decrease, with the reduction being 25% of t o t a l possible at a 1:1 r a t i o . This i s s i g n i f i c a n t l y less than the >80% decrease brought about by a 1:1 r a t i o of DNase I/TM under s i m i l a r s a l t conditions (Payne et al., 1986). It can be seen that the addition of ImM C a 2 + to TM alone causes a decrease i n T i r e l that i s greater than expected due to a 1% d i l u t i o n e f f e c t . This e f f e c t of bivalent cations on TM v i s c o s i t y i s well documented (Ooi et al., 1962; I s h i i and Lehrer, 1989). 4.2.1. Fluorescence Studies Dansyl chloride(DNS)-labelled C-TM (12 DNS per C-TM) i n 150mM KC1, 25mM Mops, pH 7.5, was sensitive to addition of g e l s o l i n (+Ca2+) . The fluorescence i n t e n s i t y of DNS-C-TM at 510nm increased 23% i n the presence of g e l s o l i n , i n d i c a t i n g that the dansyl groups bound to Lys residues on the surface of the C-TM are s h i f t e d to a more non-polar environment i n the presence of g e l s o l i n . The fluorescence p o l a r i z a t i o n increased from 0.124 to 0.192, consistent with slower r o t a t i o n a l motion of the complex r e l a t i v e to TM alone, possibly due simply to i t s greater s i z e . There are 15 Trp residues assumed to be i n horse plasma g e l s o l i n (Ruiz S i l v a and Burtnick, 1990) as t h i s number i s conserved i n p i g and human plasma g e l s o l i n (Way and Weeds, 87 Log [Gelsolin](M) FIGURE 31. Ef f e c t of g e l s o l i n on the low i o n i c strength polymerizability of C-TM (1 x 10~5M) . Relative v i s c o s i t i e s of solutions containing d i f f e r e n t amounts of g e l s o l i n were measured i n 2mM T r i s , 2mM DTT, 0.2mM CaCl 2, 0.4mM EDTA, pH 7.6 (•), and then remeasured a f t e r the addition of ImM CaCl 2 (O). T = 27°C. 88 1988; Kwiatkowski et al., 1986). Quenching of g e l s o l i n Trp fluorescence was c a r r i e d out i n the absence and presence of a 2-fold molar excess of tropomyosin i n 150mM KC1, 20mM Mops, 4mM CaCl 2, pH 7.0, to determine i f the in t e r a c t i o n affected the access of solvent to the tryptophans. Figure 32 shows that i n the presence of tropomyosin, the Trp fluorescence i s quenched less e f f e c t i v e l y than i n i t s presence. Without knowledge of the structure of g e l s o l i n and where the Trp residues are located, i t i s only possible to conclude that the in t e r a c t i o n with tropomyosin r e s u l t s i n protection of the tryptophans either d i r e c t l y by physical s h i e l d i n g or i n d i r e c t l y by inducing a conformational change i n the g e l s o l i n . Acrylodan p o l a r i z a t i o n values from AD-C-TM and AD-P-TM i n 150mM KC1, 20mM Mops, 4mM CaCl 2, pH 7.0, were e s s e n t i a l l y unchanged i n the absence and presence of g e l s o l i n , as were the slopes of the Stern-Volmer quenching p l o t s . This suggests that the Cys regions on the TMs are not affected by the i n t e r a c t i o n with g e l s o l i n , i n marked contrast to the int e r a c t i o n with DNase I. When g e l s o l i n was l a b e l l e d with acrylodan (degree of l a b e l l i n g = 5 AD per g e l s o l i n ) , there was no e f f e c t on the emission c h a r a c t e r i s t i c s of the acrylodan i n the presence of tropomyosin. This suggests that the acrylodan l a b e l l i n g s i t e s were not near the s i t e that i n t e r a c t s with tropomyosin. There was a small reduction i n the p o l a r i z a t i o n of the AD fluorescence when Ca 2 + was added to g e l s o l i n alone (0.266 —> 0.252), i n d i c a t i v e of a 89 loosening of the molecule i n the v i c i n i t y of the l a b e l l i n g s i t e (s). FIGURE 32. Stern-Volmer plots for iodide quenching of tryptophan fluorescence from g e l s o l i n (1UM) i n the absence (•) and presence (•) of C-TM (2uM) i n 150mM KC1, 20mM Mops, 4mM CaCl 2/ pH 7 . 0 . 5. CONCLUSIONS The main conclusion from Chapter 3 is that the COOH-terminus of TM i s required to hold the COOH-terminal half of the molecule, the Cys-190 region in particular, in a specific conformation. The effect of removal of a few amino acid residues from the COOH-terminal is readily detectable at Cys-190, the site of attachment of pyrene and acrylodan labels used in this study, some 90 amino acids away. The difference in the overall structure between C-TM and NPTM i s small but the unique information available as a result of excimer formation between pyrenes at Cys-19.0 on adjacent chains suggests that a change occurs in the interchain relationship in this region upon removal of the COOH-terminus. This long-range communication between distant regions on TM could well influence how TM participates in regulation of striated muscle contraction. Our results show that this communication can occur independently of the binding of TM to F-actin or to troponin T, which is thought to interact with TM at sites near to Cys-190 and at the COOH-terminal of TM (Mak & Smillie, 1981b). The findings from studies on the hybridization of C-TM with NPTM and C-TM with P-TM support the conclusion of Brown and Schachat (1985) that renaturation of denatured tropomyosin depends on i n i t i a l interaction in the NH2-terminal third of the molecule. C-TM and NPTM are identical except for the COOH-terminus excisions in NPTM. Cooling of a 91 denatured mixture seems to produce a s i g n i f i c a n t proportion of hybrid due to the homology of the two species. C-TM and P-TM have a high degree of homology i n the central part of t h e i r sequences but are quite d i f f e r e n t i n the NH 2-terminus region. There also i s a difference of 37 residues i n t h e i r lengths, which constitutes a p o t e n t i a l l y large d e s t a b i l i z i n g component i n the hybrid molecule. It was only possible to form the C-TM/P-TM hybrid by rapid thermal quenching to 4°C, but even then i t was produced only i n small quantities. The main conclusion from the f i r s t part of Chapter 4 i s that the in t e r a c t i o n between DNase I and tropomyosin causes l o c a l i z e d conformational changes on the tropomyosin that may explain the reduced end-to-end polymerizability i n the presence of DNase I. The red-shifted emission maximum, the decreased fluorescence i n t e n s i t y , the increased a c c e s s i b i l i t y to quenching by iodide ions and the diminished fluorescence p o l a r i z a t i o n values for each acrylodan-labelled tropomyosin i n the presence of DNase I a l l indicate increased solvent exposure and f l e x i b i l i t y of the s i t e of l a b e l l i n g . Cys-246 of P-TM i s the penultimate COOH-terminus residue and Cys-153 of truncated P-TM i s analogous to Cys-190 of C-TM. A l l three s i t e s show the same q u a l i t a t i v e changes i n the presence of DNase I suggesting a c o r r e l a t i o n between s t r u c t u r a l changes at the in t e r n a l Cys and the COOH-terminus, i n support of the findings of Chapter 3. The finding that g e l s o l i n reduces the end-to-end poly m e r i z a b i l i t y of tropomyosin makes i t another a c t i n -92 binding protein that a f f e c t s tropomyosin d i r e c t l y . The fluorescence data suggest that g e l s o l i n does not disrupt the tropomyosin structure to the same extent as does DNase I. Like DNase I, g e l s o l i n disrupts F-actin filaments (in the presence of Ca 2 +) but unlike DNase I, g e l s o l i n does t h i s by severing the filament. This severing i s i n h i b i t e d i f muscle TM i s bound to the F-actin filaments (Fattoum et al., 1983). Low molecular weight tropomyosins do not i n h i b i t the severing unless non-muscle caldesmon i s added (Ishikawa et al., 1989). Caldesmon has also been found to increase the v i s c o s i t y of smooth muscle tropomyosin when added (Graceffa, 1987). The possible trend seems to be that actin-binding proteins that s t a b i l i z e the F-actin-TM complex increase the end-to-end p o l y m e r i z a b i l i t y of TM (troponin (Ebashi and Kodama, 1965), caldesmon) and actin-binding proteins that d e s t a b i l i z e the F-actin-TM complex decrease the end-to-end p o l y m e r i z a b i l i t y of TM (DNase 1/ g e l s o l i n ) . More a c t i n -binding proteins should be tested for t h e i r e f f e c t on tropomyosin to see i f they f i t t h i s pattern. 93 6. BIBLIOGRAPHY A j t a i , K., and Venyaminov, S.Y. (1983) FEBS Lett. 151, 94-96 Bailey, K. (1946) Nature 157, 368-369 Betcher-Lange, S.L., and Lehrer, S.S. (1978) J. Biol. Chem. 253, 3757-3760 Betteridge, D.R., and Lehrer, S.S. 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APPENDIX I LIST OF SYMBOLS AND ABBREVIATIONS cc-TM A cla s s of tropomyosin chain represented by that found i n rabbit cardiac muscle. e Molar extinc t i o n c o e f f i c i e n t 4> Fluorescence quantum y i e l d T| V i s c o s i t y U Absorption dipole moment 6 E l l i p i t i c i t y X Fluorescence l i f e t i m e A Fluorescence anisotopy Absorbance at wavelength, X AcOH Ethanoic a c i d AD Acrylodan, 6-acryloyl-2-dimethylamino-napthalene ATPase Enzyme able to catalyse hydrolysis of adenosine triphosphate CD C i r c u l a r dichroism C-TM Cardiac tropomyosin C-TM* Tropomyosin species with one C-TM chain and one NPTM chain Cys Cysteine DFP Diisopropyl fluorophosphate DNase I Deoxyribonuclease I DNS-C1 Dansyl Chloride, 5-dimethylaminonaphthalene-1-sulfonyl chloride DTT D i t h i o t h r e i t o l EDTA Ethylenediamine-tetraacetic acid 100 G-TM Gizzard tropomyosin GuHCl Guanidine hydrochloride 1 , 1 In t e n s i t i e s of polarized fluorescence, p a r a l l e l and perpendicular respectively, to the polari z e d e x c i t a t i o n beam k F Fluorescence rate constant k i c Rate constant f o r in t e r n a l conversion k i s Rate constant for intersystem crossing between s i n g l e t and t r i p l e t e l e c t r o n i c l e v e l s k q Bimolecular rate constant for fluorescence quenching MeOH Methanol Mops 4- (W-morpholino)propanesulfonic acid NbS 2-nitro-5-thiobenzoate (NbS) 2 5,5'-dithiobis(2-nitrobenzoate) NPTM Non-polymerizable cardiac tropomyosin p Fluorescence p o l a r i z a t i o n PMSF Phenylmethylsulfonyl f l u o r i d e P-TM P l a t e l e t tropomyosin P-TM* P l a t e l e t tropomyosin treated with carboxypeptidase A Py Pyrene SI Enzymically prepared part of myosin molecule that binds to acti n SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis S-TM Skeletal tropomyosin TLC Thin layer chromatography Tn Troponin T r i s Tris(hydroxymethyl)methylamine Tryptophan Tyrosine Molecular volume 

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