Thermal transitions of myosin and its helical fragments. Regions of structural instability in the myosin molecule.

The structural stabilities of all the familiar proteolytic fragments of myosin have been investigated in melting studies over the pH ranges 5.5-7.0 in 0.5 M KCl. All fragments except subfragment 2 undergo a melting transition manifested by the cooperative uptake of protons in the temperature range 34-47 degrees C, and these fragments experience an increase in transition temperature, Tm as the pH is increased. Subfragment 2 undergoes a melting transition in the 43-55 degrees C range, manifested by the dissociation of protons, and it experiences a decrease in Tm as the pH is increased. These results suggest that pH changes can modulate the relative stabilities of the light meromysin, subfragment-1, and subfragment-2 regions of the myosin molecule.     

Thermal transitions of myosin and its helical fragments. II. Solvent-induced variations in conformational stability.

The melting behavior of myosin and myosin rod has been studied over the pH range 5.4-7.0, in 0.5 M KCl. Both proteins exhibit almost identical T-m values, which increase from about 37 to 43 degrees as the pH is elevated through the range of study, T-m follows a sigmoidal dependence upon pH, and the inflection point occurs near pH 6.5. The influence of salt concentration on T-m was studied, by the variation of KCl from 0.15 to 2.92 M. With an increasing KCl concentration, both proteins exhibit similar, sigmoidal declines in T-m from about 44 to 34 degrees. Under all conditions of pH and ionic strength studied, melting is accompanied by an absorption of H+ by the protein. The concentration of the protein, the age of the preparation, and the presence of divalent metal ions fail to exert a significant effect on the T-m values obtained by our methods. The effects of salt concentration and pH on the thermal stability of myosin and myosin rod are discussed in terms of the location of the melting process within the myosin molecule. Myosin is shown to possess several of the requisite features for energy transduction via a proton-coupling mechanism. These features provide a framework for investigating some aspects of the molecular basis of muscle contraction within the context of the sliding filament model.     

Thermal stability of myosin rod from various species

The radius of gyration and fraction helix as a function of temperature have been determined for myosin rod from four different species: rabbit, frog, scallop, and antarctic fish. Measurements from sodium dodecyl sulfate gel electrophoresis indicate that all particles have the same molecular weight (approximately 130K). All fragments are nearly 100% alpha-helical at low temperatures (0-5 degrees C). The melting profiles for each are qualitatively similar in shape, but their midpoints are shifted along the temperature axis in the following order: antarctic fish (Tm = 33 degrees C), scallop (Tm = 39 degrees C), frog (Tm = 45 degrees C), and rabbit (Tm = 49 degrees C). Corresponding radius of gyration vs temperature profiles for each species are shifted to lower temperatures (approximately 5-8 degrees C) with respect to the optical rotation melting curves. From plots of radius of gyration vs fraction helix, we find a marked drop in the radius of gyration (from 43 to approximately 34 nm) with less than a 5% decrease in fraction helix for rabbit, frog, and antarctic fish rods, whereas the radius of gyration of scallop rod never exceeds 34 nm. Results indicate hinging of the myosin rod of each species. The thermal stabilities of the myosin rods shift in parallel with the working temperature of their respective muscles.     

Study of effects of pH on the stability of domains in myosin rod by high-resolution differential scanning calorimetry

Differential scanning calorimetry (DSC) has detected at least six quasi-independent structure domains in myosin rod [Potekhin, S.A., & Privalov, P.L. (1978) Biofizika 23, 219-223]. These domains were found to be remarkably sensitive to pH in the physiological range, i.e., pH 6-8. We compared the thermodynamic characteristics, and studied effects of pH on the stability, of individual domains in rod, light meromyosin (LMM), and subfragment 2 (S-2). In rod, the lowest stability domain (approximately 400 amino acid residues per double strand), with a Tm of 42.4 degrees C, a delta Hcal of 190 kcal/mol, and a delta G of 3.39 kcal/mol, at pH 7.02, destabilized by absorption of protons, is located at the LMM/S-2 junction and split into two parts, one associated with S-2 (approximately 100 residues per double strand) and the other with LMM (300 residues per double strand). The fragment with S-2 is likely a part of the "hinge" suggested by Swenson and Ritchie [(1980) Biochemistry 19, 5371-5375]. All other domains of rod released protons on melting. The domains located in S-2 were the most sensitive to pH and released a total of 0.9 proton on melting. The thermal meltings of all domains in myosin rod, LMM, and S-2 were independent of each other, and enthalpies of melting were additive in the whole pH range studied. Their sensitivities to pH and KCl were also unaffected by the presence or absence of other fragments. For example, domains in an isolated S-2 behaved similarly as they were in the rod, and so were domains in LMM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rigidity of myosin and myosin rod by electric birefringence

The rotational relaxation times of rabbit myosin and myosin rod have been determined by electric birefringence measurement. The relaxation time of myosin measured in 10 mM pyrophosphate buffers in a pH range of 7.6–9.5 was found to have substantial concentration and pH dependences. The infinite-dilution limit of the relaxation time, τ°, was determined as 38 ± 2 μs, and it was found to be independent of pH. For myosin rod, a possible thermally induced conformational change was investigated in a temperature range of 1–43°C. The rotational relaxation time of myosin rod shows no clear indication of conformational change in this temperature range, and the radius of gyration measurement by light scattering was shown to be consistent with this observation. The steady-state birefringence, however, decreases substantially above around 40°C. This, the myosin rod appears to be only slightly flexible even at physiological temperature, but the possibility of a “melting” or “hinging” of the myosin rod cannot completely be ruled out on the basis of these experiments.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Binding of ampholine to transfer RNA.

The melting temperature of isoaccepting tRNAfMet is affected by Ampholine. The plot of Tm versus the logarithm of Ampholine concentration shows clearly an increasing effect of Ampholine when the pH changes from 7.4 to 4.2. This result is interpreted as binding of Ampholine to the nucleic acid. The effects of Ampholine have been compared with those of soidum, magnesium and tetraethylene pentamine. Ampholine carrier ampholytes at pH 4.2 bind to tRNA with the same affinity as magnesium; at higher pH values they are less active. An hypothesis for the mechanism of action of Ampholine on nucleic acids during isoelectric focusing is proposed.     

An enzyme-probe method to detect structural changes in the myosin rod

The temperature-dependence of local melting within the alpha-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (alpha-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 degrees C (pH 7, I = 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 degrees C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 degrees C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.     

Effect of various anions on the stability of the coiled coil of skeletal muscle myosin

The stability of skeletal myosin rod was studied by following the dependence of both papain digestion kinetics and helix-coil transition temperatures on the concentration of neutral salts. The rate of papain-catalyzed digestion of rod to form subfragment 2 and light meromyosin was strongly dependent on chloride concentration but essentially independent of acetate concentration up to 2.0 M. The rod exhibited a biphasic melting curve in 0.6 M NaCl, 5 mM phosphate, and 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.3, with transitions at 45 and 53 degrees C. In 0.6 M CH3COONa, 5 mM phosphate, and 0.1 mM EDTA, pH 7.3, the transitions occurred at 50 and 58 degrees C, respectively. Transition temperatures were obtained with a novel curve-fitting method. The effect of increasing chloride ion concentration on melting profiles was 2-fold. Below 0.6 M salt, the two transition temperatures, Tm,1 and Tm,2, depended on salt concentration such that increasing NaCl concentration caused a small stabilization of the helix while increasing acetate concentration caused the helix to become markedly more stable. Between 0.6 and 1.0 M, variation of chloride concentration had almost no effect on the thermal stability of the rod while increasing acetate concentration increased its stability considerably. Above 1.0 M NaCl, the melting profiles became broad with a third transition being observed (e.g., at 3.0 M, Tm,3 = 38 degrees C), indicating the existence of a region which has a tendency to be destabilized by chloride. The third transition was not observed at comparable concentrations of acetate. This effect of chloride was not expected on the basis of its position in the Hofmeister series.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on interaction between poly(L-lysine58, L-phenylalanine42) and deoxyribonucleic acids.

A random copolymer of 58% L-lysine and 42% L-phenylalanine, poly(Lys58Phe42), was used as a model protein for studying the role of phenylalanine residues in protein-DNA interaction. Complexes between this copolypeptide and DNA, made by direct mixing, were studied by absorbance, circular dichroism (CD), fluorescence, and thermal denaturation. Complex formation results in an increase in absorbance, and an enhancement, red-shift, and broadening of phenylalanine fluorescence. The fluorescence enhancement is opposite to the quenching observed when a tyrosine copolypeptide is bound to DNA (R. M. Santella and H.J. Li (1974), Biopolymers 13, 1909). The positive CD band of DNA near 275 nm is reduced and red-shifted by the binding of the phenylalanine copolypeptide to a greater extent than by the tyrosine copolypeptide. Thermal denaturation of the complexes in 2.5 times 10(-4) M EDTA (pH 8.0) shows three characteristic melting bands. For complexes with calf thymus DNA, free base pairs melt at Tm,I (47-49 degrees) and copolypeptide-bound base pairs show two melting bands (Tm,II at 73-75 degrees, and Tm,III at 88 -90 degrees). Similar thermal denaturation results have been observed for complexes with Micrococcus luteus DNA. The fluorecence intensity of the complexes is greatly increased when the temperature is raised to the Tm,II region. In addition to fluorescence measurements, the effects of increasing temperature on absorption and CD spectra of the complexes were also studied. Stacking interaction between the phenylalanine chromophore and DNA bases, either partial or full intercalation, is implicated by the experimental results. Several mechanisms are proposed to describe the reaction between the copolypeptide and DNA, and thermal denaturation of the complex.     

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle contraction.     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.     

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Ultra short yeast tropomyosins show novel myosin regulation

Tropomyosin (Tm) is an alpha-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K(50%) approximately 0.5 microm). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosin-binding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model.     

Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

Force spectroscopy reveals multiple "closed states" of the muscle thin filament

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.     

Thermodynamics of maltose binding protein unfolding.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.