Smooth muscle tropomyosin coiled-coil dimers. Subunit composition, assembly, and end-to-end interaction

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.     

In-register homodimers of smooth muscle tropomyosin

Gizzard smooth muscle tropomyosin dimer molecules were dissociated by guanidinium chloride and reassociated by dialysis against 1 M NaCl. Several properties of the protein were changed by this treatment. There was a large decrease in tropomyosin's low-salt viscosity, owing to reduced end-to-end polymerization, the helix unfolding profile changed from a one-step to a two-step process, and the ability to form intramolecular, interchain, disulfide-cross-linked homodimers increased dramatically. Thus, the native molecule, though to exist predominantly as by the beta gamma heterodimer which cannot form disulfide cross-links [Sanders, C., Burtnick, L.D., & Smillie, L. B. (1986) J. Biol. Chem. 261, 12774-12778], reassembles, after dissociation, to form predominantly parallel, in-register beta beta and gamma gamma homodimers able to form disulfide cross-links. This suggests that the physical properties, including the end-to-end interaction, of gizzard tropomyosin homodimers differ considerably from those of the heterodimer. This is a first step toward a molecular understanding of the end-to-end interaction of smooth muscle tropomyosin.     

Functional homodimers and heterodimers of recombinant smooth muscle tropomyosin

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers. We show that (1) AS alpha-Tm binds actin 100-fold tighter than alpha-Tm and 2-fold tighter than native smooth alphabeta-Tm, (2) beta-Tm requires an AS extension to bind actin, and (3) AS beta-Tm binds actin 10-fold weaker than AS alpha-Tm. Tm is present in smooth muscle tissues as >95% heterodimer; therefore, we studied the binding of recombinant alphabeta heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both alpha and beta chains to bind like native Tm and that the alpha chain contributes more to actin binding than the beta chain. Once assembled onto an actin filament, all smooth muscle Tm's regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Fluorescence from pyrene-labeled native and reconstituted chicken gizzard tropomyosins

Sulfhydryl groups at Cys-36 on the beta chain and at Cys-190 on the gamma chain of chicken gizzard tropomyosin were reacted with the pyrene-containing sulfhydryl-specific reagents N-(1-pyrenyl)iodoacetamide and N-(1-pyrenyl)maleimide. Tropomyosin prepared and labeled under nondenaturing conditions displayed significant pyrene monomer emission but low levels of pyrene excimer fluorescence. In contrast, tropomyosin subjected to denaturation and renaturation prior to labeling, or labeled in the denatured state prior to renaturation, displayed considerable excimer emission. Furthermore, labeling of isolated beta or gamma chains in denaturant, followed by reconstitution, gave separate samples of beta beta- and gamma gamma-tropomyosin that exhibited even greater pyrene excimer to monomer emission ratios. As pyrene excimers can form only when an excited pyrene is immediately adjacent to a ground state pyrene, i.e., when the labeled Cys residues on the two chains in a tropomyosin coiled coil share the same cross section, these results support conclusions based upon chemical crosslinking studies [C. Sanders, L. D. Burtnick, and L. B. Smillie (1986) J. Biol. Chem. 261, 12774-12778] that native gizzard tropomyosin exists predominantly as a beta gamma-heterodimer. In addition, the low degree of labeling of native gizzard tropomyosin and the differences in degrees of labeling of beta beta- and gamma gamma-tropomyosins in the absence of denaturants reflect on the accessibilities of the sulfhydryl groups in these tropomyosin isoforms. Circular dichroism measurements indicate that the labeled proteins form stable coiled coil structures that have thermal stabilities comparable to that of the native protein.     

Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties

A method for the rapid purification of caldesmon, an F-actin binding protein of smooth muscle, has been developed. Caldesmon remains native after heating at 90 degrees C, a property that provides the basis for the purification in high yield of both caldesmon and tropomyosin, another heat-stable protein of smooth muscle. Caldesmon purified by this procedure is a highly asymmetric protein with a sedimentation coefficient of approximately 2.7 S and a Stokes radius of about 91 A. The protein exists as two polypeptide chains of Mr = 135,000 and 140,000, with each Mr polypeptide being resolvable into several isoelectric species. Estimates based on densitometry of stained gels suggest that caldesmon is more abundant in smooth muscle than filamin or alpha-actinin. Purified caldesmon bound to F-actin in the pH range 6-8. Binding was unaffected by Ca2+ or Mg2+ at up to millimolar levels. Binding was saturable, with a polypeptide molar ratio of about one caldesmon to six actins at saturation. F-actin binding was not inhibited by saturating levels of tropomyosin. Caldesmon dramatically increased the viscosity of F-actin. Light microscopy and electron microscopy of negatively stained material revealed that caldesmon induced the formation of massive F-actin bundles which contained up to hundreds of filaments. Electron microscopy of sectioned caldesmon-saturated F-actin mixtures revealed large bundles which appeared to include linear arrays of regularly spaced actin filaments cut transversely, exhibiting a center to center spacing of 15 nm. Possible structural implications based on the existence of these structures is presented.     

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)     

In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein

CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies. Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4. The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit.     

Unfolding/refolding studies of smooth muscle tropomyosin. Evidence for a chain exchange mechanism in the preferential assembly of the native heterodimer

The thermal and the urea-induced unfolding profiles of the coiled-coil alpha-helix of native and refolded tropomyosin from chicken gizzard were studied by circular dichroism. Refolding of tropomyosin at low temperature from alpha + beta subunits, dissociated by guanidinium chloride, urea, or high temperature, predominantly produced alpha alpha + beta beta homodimers in agreement with earlier studies of refolding from guanidinium chloride (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The presence of two unfolding transitions in low salt solutions with about equal helix loss verified the composition with the first unfolding transition of the homodimer mixture originating from alpha alpha. In contrast, refolding by equilibrating at temperatures close to physiological, however, produced the native alpha beta heterodimer, which unfolded in a single transition. The refolding kinetics of dissociated alpha + beta subunits indicated that beta beta homodimers form first, leading to alpha alpha homodimers both of which are relatively stable against chain exchange below approximately 25 degrees C. Equilibrating the homodimer mixture at 37-40 degrees C for long times, however, produced the native alpha beta molecule via chain exchange. The equilibria involved indicate that the free energy of formation from subunits of alpha beta is much less than that of (alpha alpha + beta beta)/2. In vivo folding of alpha beta from the two separate alpha and beta gene products is, therefore, thermodynamically favored over the formation of homodimers and biological factors need not be considered to explain the native preferred alpha beta composition.     

Tropomyosin's end-to-end polymerization is irreversibly lost on exposure to urea or cyanate

Tropomyosin's low-salt viscosity which is due to end-to-end polymerization, is irreversibly lost upon incubation in 8M urea at room temperature. This effect is due to the chemical modification of lysine residues by cyanate in the urea. In the absence of urea, cyanate alone has the same effect. A loss in tropomyosin binding to actin accompanies the loss in viscosity, consistent with the view that tropomyosin's end-to-end interaction is necessary for strong binding to actin. During column chromatography in 8M urea, used to separate the alpha and beta chains of tropomyosin, the loss of viscosity can be minimized by using freshly-prepared urea and by reducing the time during which the protein and urea are in contact.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.     

Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.     

Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).     

Flexibility of smooth and skeletal tropomyosins

The rotational relaxation times of nonpolymerizable skeletal and smooth muscle tropomyosin were measured by analysis of the decay of the zero-field birefringence at different temperatures and salt concentrations. Skeletal tropomyosin in solution is equally well modeled as a rigid rod or as a semiflexible rod with a persistence length of 150 nm. Smooth muscle tropomyosin does not fit the rigid rod model but is well approximated by a semiflexible rod model with a persistence length of 55 nm. The results indicate that smooth muscle tropomyosin is either a more flexible molecule than skeletal muscle tropomyosin or is a curved structure with an end-to-end length shorter than the coiled-coil contour length. Smooth muscle tropomyosin controls the actomyosin ATPase differently from skeletal muscle tropomyosin and it had been suggested that the reason is because it is more rigid; clearly, another explanation must be sought.     

Isolation and characterization of tropomyosin kinase from chicken embryo

Tropomyosin kinase is partially purified from 14-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of Mr = 76,000 and 43,000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250,000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent Km for skeletal alpha-tropomyosin and ATP is 50 microM and 200 microM, respectively. Vmax varies between 100-300 nmol/min per mg depending on the purity of the preparation. Mg2+ and dithiothreitol are essential for activity but Ca+, calmodulin and cAMP are not required. The optimum temperature is 37 degrees C and optimum pH is about 7.5. Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

Structural analysis of human skeletal beta-tropomyosin produced in E. coli

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.     

Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...