The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.     

Quantitative analysis of tropomyosin linear polymerization equilibrium as a function of ionic strength.

Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken alpha-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin.     

A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units

Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding. In contrast, under high pressure, unlabeled and labeled tropomyosin molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.     

Poly(2-amino-8-methyladenylic acid). Competing structural and energetic effects of substituents

The ribopolynucleotide poly(2-amino-8-methyladenylic acid), (r2NH2(8)MeA)n, has been synthesized, and its physical and chemical properties have been examined. The study reveals competing effects on these properties of the 2-NH2 and 8-Me substituents. In marked contrast to the analogous (r8MeA)n, the new polymer readily interacts to form double helixes with complementary pyrimidine polynucleotides. Triple helixes are not formed. The 8-Me group is strongly destabilizing for helix formation (delta Tm approximately 65 degrees C), presumably by favoring a syn conformation, which blocks heteroduplex formation with ribohomopolymers. The 2-NH2 substituent stabilizes helixes in the ribo series by about 30 degrees C in Tm by forming a third interbase hydrogen bond. We suggest that the free energy from the 2-NH2 interaction drives the syn-anti equilibrium to the purine polymer to the anti form present in the double helix. CD spectra of the homopolymers (r2NH2A)n and (r2NH2(8)MeA)n are completely different, reflecting major differences of conformation. The double helixes formed by these polymers with (rT)n and (rBrU)n, on the other hand, have closely similar CD spectra, supporting our proposal of a major change in conformation of (2NH2(8)MeA)n on going from single strand to double helix.     

Alpha-helix to random coil transitions of two-chain coiled coils: experiments on the thermal denaturation of isolated segments of alpha alpha-tropomyosin

Circular dichroism (CD) experiments in the backbone (200-240 nm) region are reported for four isolated, excised two-chain, coiled-coil segments whose chains comprise, respectively, residues 11-127, 142-281, 1-189, and 190-284 of the rabbit alpha alpha-tropomyosin (Tm) sequence. The uv and CD spectra for the noncross-linked segments are very similar to those for parent Tm. At 3 degrees C, all have a helix content of 90% or more; moreover, all thermal denaturation curves depend on concentration, as required by mass action, and are completely reversible. At comparable concentrations, solutions show values of T1/2 (the temperature at which the helix content is 50%) following the order of 11Tm127 approximately 1Tm189 greater than 142Tm281 greater than 190Tm284. The thermal unfolding data for 11Tm127, 190Tm284, and 142Tm281 fall on apparently monophasic curves (single inflection point). However, curves for 1Tm189 show a heretofore unknown low temperature transition in which the helix content drops from approximately 90% at 2 degrees C to approximately 73% at 20 degrees C, indicating that this segment has one or more weak sections totaling approximately 50 residues per chain. Since thermal denaturation curves for noncross-linked 11Tm127, 142Tm281, and Tm have no such low temperature transition, i.e., the helix content is not additive, the weak region probably comprises the bulk of the residues between 127 and 189 in 1Tm189, but is somehow stabilized in 142Tm281 and in parent Tm.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some properties of acid-reassembled tropomyosin

The preference for parallelism of the two chains in tropomyosin coiled coils is thought to result from interchain salt bridges. To examine this idea, studies are presented of tropomyosin molecules reassembled from chaotropic solvents in acid solution, where cross-links cannot exist. The acid-reassembled molecules are appreciably less disulfide cross-linkable in acid than native molecules, a result explainable if some antiparallel dimers indeed form at low pH. Physical studies (backbone- and tyrosine-region CD and intrinsic viscosity) indicate that refolding in acid yields a molecular population demonstrably different in tyrosine-region CD from native, but having comparable (but not identical) helix content, thermal stability, and dimensions. Moreover, the refolding in acid after either thermal or chaotropic-solvent denaturation yields the same final state, arguing that it is an equilibrium state. All these results are consistent with, but do not prove, that the acid-reassembled population includes an appreciable fraction (2/3) of antiparallel coiled-coil dimers. © 1995 John Wiley & Sons, Inc.     

Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex

Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tm head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head-to-tail complex stability by modulating the stability of the helices at the Tm termini.     

Diffusion and partitioning of proteins in charged agarose gels.

The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.     

Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase.

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.     

The effect of single residue substitutions of serine-283 on the strength of head-to-tail interaction and actin binding properties of rabbit skeletal muscle alpha-tropomyosin

Vertebrate skeletal muscle alpha-tropomyosin polymerizes in a head-to-tail manner and binds cooperatively to actin. It has been postulated that the cooperative actin binding is governed by the strength of the head-to-tail interaction. In order to know the relationship between the head-to-tail affinity and actin binding, we studied the properties of tropomyosin variants with single residue substitutions at serine-283, the penultimate residue at the carboxyl terminus that is involved in the head-to-tail interaction. It has been shown that the phosphorylation of serine-283 strengthens the head-to-tail interaction. Viscometry was employed to compare the head-to-tail affinity of tropomyosin variants. Variant S283E showed higher viscosity whereas variant S283K showed lower viscosity compared with the wild type non-phosphorylated alpha-tropomyosin. The results confirm the idea that the interaction is sensitive to the ionic properties of residue 283. The strength of the head-to-tail interaction was assessed directly by sedimentation equilibrium using two pairs of tropomyosin variants designed so that only dimeric interactions were allowed within each pair. From one pair of variants with serine-283, the association constant was determined to be 2.6 x 10(4) M(-1) (SD =1.0 x 10(4)), whereas for the second pair with glutamate-283, the affinity was 3.9 x 10(4) M(-1) (SD =1.6 x 10(4)), slightly stronger than the former, consistent with the results of viscometry. The results indicate that the head-to-tail association is weak as previously implicated from light scattering measurements. Cosedimentation was employed to measure the cooperative actin binding of tropomyosin variants. Although previous results indicated the phosphorylation has no significant influence on the actin affinity, variant S283E shows a lower affinity compared with the control. Variants S283K and S283A show even lower affinities to actin, although these species bind to actin more cooperatively than does variant S283E. The results indicate that the affinity of the head-to-tail interaction between adjacent tropomyosin molecules is weak, and is substantially influenced by an extra charge at residue 283. On the other hand, the interaction with actin, the affinity and the cooperativity in actin binding, is dependent on amino acid residues at 283 and is not simply correlated with the strength of the head-to-tail interaction between Tm molecules in solution.     

Crosslinking of actin filaments and inhibition of actomyosin subfragment-1 ATPase activity by streptococcal M6 protein

M proteins are antiphagocytic molecules on the surface of group A streptococci having physical characteristics similar to those of mammalian tropomyosin. Both are alpha-helical coiled-coil fibrous structures with a similar seven-residue periodicity of nonpolar and charged amino acids. To determine if M protein is functionally similar to tropomyosin we studied the interaction of M protein with F-actin. At low ionic strength, M protein binds to actin weakly with a stoichiometry different from that of tropomyosin. M protein does not compete with tropomyosin for the binding to actin, indicating that it is functionally different from tropomyosin. M protein does compete with myosin subfragment-1 for binding to actin and induces the formation of bundles of actin filaments. The formation of actin aggregates is associated with a sharp reduction in the rate of ATP hydrolysis by subfragment-1. Intact streptococci having M protein on their surface are shown to bind to actin.     

Comparison of effects of smooth and skeletal muscle tropomyosins on interactions of actin and myosin subfragment 1

The ATPase activity of acto-myosin subfragment 1 (S-1) was measured in the presence of smooth and skeletal muscle tropomyosins over a wide range of ionic strengths (20-120 mM). In contrast to the 60% inhibitory effect caused by skeletal muscle tropomyosin at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on ionic strength. At low ionic strength (20 mM), smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM), it potentiates the ATPase activity 3-fold. All of these ATPase activities were measured at very low ratios of S-1 to actin, under conditions at which a 4-fold increase in S-1 concentration did not change the specific activity of the tropomyosin-acto.S-1 ATPase. Therefore, the potentiation of the ATPase activity by smooth muscle tropomyosin at high ionic strength cannot be explained by bound S-1 heads cooperatively turning on the tropomyosin-actin complex. To determine whether the fully potentiated rates are different in the presence of smooth muscle and skeletal muscle tropomyosins, S-1 which was extensively modified by N-ethylmaleimide was added to the ATPase assay to attain high ratios of S-1 to actin. The results showed that, under all conditions, the fully potentiated rates are the same for both tropomyosins.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD and (13)C(alpha)-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of alpha-tropomyosin

Synthesis and CD and (13)C(alpha)-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide ((190)Tm(254)) comprising residues 190-254 of the alpha-tropomyosin chain. CD on a version disulfide cross-linked via the N-terminal cysteine side chains indicates that this dimer is highly helical and melts near 48 degrees C. The CD is independent of peptide concentration, showing that association of (190)Tm(254) stops at the two-strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27 degrees C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates (190)Tm(254) as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for (190)Tm(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for (190)Tm(254) also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others--such as the destabilizing effect of alanine in d, the dimer-disfavoring effect of beta-branching in d and its dimer-favoring effect in a, and the dimer-directing effect of asparagine in a--are more questionable in tropomyosin than in the leucine zipper coiled coils. (13)C(alpha)-NMR data at two labeled sites, L228(d) and V246(a), of (190)Tm(254) display well-separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.     

Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification

The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K(T)) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys. J. 74, A347]. The data could be accounted for by changes in K(T) (0.1-1) with no change in n. Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction. These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties.