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.     

Preferential assembly of the tropomyosin heterodimer: equilibrium studies

Thermal unfolding/refolding studies of the three tropomyosin dimers, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into heterodimers, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. 265, 1134]. Circular dichroism measurements showed that all three dimers unfolded in cooperative reversible transitions with T1/2 = 40.0 degrees C and delta H degrees = 162 kcal/mol for alpha alpha and with T1/2 = 42.6 degrees C and delta H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homodimers, (ii) at physiological temperature, heterodimers are formed from micromolar mixtures of homodimers over a period of minutes, and (iii) heterodimers unfold/refold with temperature without appreciable formation of homodimers. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for delta H degrees = 225 kcal/mol and T1/2 = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.     

In vitro phosphorylation of tropomyosin by a kinase from chicken embryo

A tropomyosin kinase has been partially purified from the leg muscle of 11-day-old chick embryos by ammonium sulfate precipitation and DEAE and phosphocellulose chromatography. The tropomyosin kinase requires Mg2+ for its activity, but Ca2+ and cyclic AMP are not needed. Increase in KC1 concentration decreased the tropomyosin kinase activity with over 90% inhibition at 0.2 M KC1. The alpha-tropomyosin subunit from rabbit and chicken skeletal muscle was phosphorylated about five times faster than the beta-tropomyosin subunit. Smooth muscle tropomyosin from chicken gizzard was not phosphorylated. The in vitro phosphorylation site in rabbit and chicken skeletal tropomyosins is a single serine residue close to the COOH terminus, a region intimately engaged in the head to tail polymerization of tropomyosin. Since the amino acid sequences of rabbit alpha- and beta-tropomyosin and chicken alpha-tropomyosin in this region are known, their phosphorylation sites can be unambiguously assigned as the penultimate residue, serine 283.     

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.     

Thermal unfolding equilibria in homodimeric chicken gizzard tropomyosin coiled coils

CD studies are presented on thermal unfolding of coiled-coil homodimers of two genetic variant chains of chicken gizzard tropomyosin (CG-Tm). The experiments include the effects of cross-linking both isoforms and the dependence on protein concentration of unfolding in both reduced isoforms, variables not examined in extant work. The general shapes of the unfolding curves for singly cross-linked species depend on whether the crosslink is at C190 (its site on one isoform) or at C36 (its site on the other). These curves are compared with extant ones for various cross-linked species of rabbit tropomyosin. The comparison supports the view that the unfolding behavior of cross-linked species results from a complex interaction of strain at the cross-link, local variations in structural stability, and loop entropy. The observed concentration dependence of the transition temperature for the uncross-linked (reduced) species of CG-Tm is very small (2.9°C) for one variant homodimer and unobservably small (< 2°C) for the other in the 100-fold concentration range (～ 0.01–1.0 mg/mL) accessible here. These experimental values of ΔT_m are much smaller than are predicted from extant values of the van't Hoff transition enthalpies, calling the latter into question. © 1994 John Wiley & Sons, Inc.     

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.     

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.     

Thermodynamics of unfolding of beta-trypsin at pH 2.8

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Calorimetric and spectroscopic investigation of drug-DNA interactions. I. The binding of netropsin to poly d(AT)

We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

High- and low-temperature unfolding of human high-density apolipoprotein A-2.

Human plasma apolipoprotein A-2 (apoA-2) is the second major protein of the high-density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid-free apoA-2 in neutral low-salt solutions is most stable at approximately 25 degrees C and unfolds reversibly both upon heating and cooling from 25 degrees C. High-temperature unfolding of apoA-2, monitored by far-UV CD, extends from 25-85 degrees C with midpoint Th = 56 +/- 2 degrees C and vant Hoff's enthalpy delta H(Th) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-helical structure. This suggests low-cooperativity apoA-2 unfolding. The apparent free energy of apoA-2 stabilization inferred from the CD analysis of the thermal unfolding, delta G(app)(25 degrees) = 0.82 +/- 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low-temperature stability of apoA-2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low delta G(app)(25 degrees), indicates that lipid-free apoA-2 has a substantial hydrophobic core but is only marginally stable under near-physiological solvent conditions. This suggests that in vivo apoA-2 transfer is unlikely to proceed via the lipid-free state. Low delta H(Th) and low apparent delta Cp approximately 0.52 kcal/mol.K inferred from the far-UV CD analysis of apoA-2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near-UV CD, are consistent with a molten globular-like state of lipid-free apoA-2.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).     

Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies

The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM ethylenediaminetetraacetic acid, a reference state. The enthalpies were measured in two buffers with different heats of ionization to allow correction for dissociation or uptake of protons. The enthalpies corrected for proton effects are -22.1, -25.4, and -23.5 kcal/mol, respectively, in buffers A, B, and C. The interaction between troponin and tropomyosin in the presence of calcium is accompanied by release of 0.9 mol of proton per mole of complex. Proton effects in the presence of magnesium and in the absence of divalent metal ions were too small to quantitate. The association constants were measured by using tropomyosin labeled with the extrinsic fluorescent probe dansylaziridine, and binding was detected by enhancement of the probe fluorescence. The magnitudes of the association constants for unlabeled troponin are 7.5 X 10(5), 4.2 X 10(5), and 9.5 X 10(5) M-1, respectively, for the three solution conditions corresponding to unitary free energies of -10.4, -10.1, and -10.6 kcal/mol. The unitary entropies for the interaction are -39, -51, and -43 cal/(deg X mol), respectively, for the three solution conditions. Under these conditions, the troponin-tropomyosin interaction is enthalpy driven, and a large unfavorable entropy must be overcome in the formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)