Estimation of denaturation of actin in the actin-myosin complex treated with various conditions by DNAase I inhibition assay.

1. Experiments were conducted to evaluate whether DNAase I (EC 3.1.4.5) inhibition assay was a valuable tool to study the denaturation of actin in the actin-myosin complex treated with various conditions. 2. A sample containing F-actin or natural actomyosin(myosin B) was treated with KI-ATP solution to convert a form which inhibits DNAase I as effectively as G-actin, and the total amount of native actin was determined by DNAase I inhibition assay. 3. On the basis of the values for remaining native actin in the sample obtained by this assay, a percentage of denaturation of actin during treatment was calculated. 4. The present result demonstrated that DNAase I inhibition assay was easy to perform, very sensitive (0.5-2.0 microgram actin) and highly specific for estimating denaturation of actin in the actin-myosin complex treated with heat or high salt concentrations. 5. In addition, the use of DNAase I and standard G-actin preparations stored frozen at -80 degrees C for the assay was found to be possible within a fixed period of time (about 2 weeks), which was helpful in monitoring the denaturation process of actin treated under various conditions for a long period.     

Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin.

The interaction of heat shock protein with molecular mass 25 kDa (HSP25) and its point mutants S77D + S81D (2D mutant) and S15D + S77D + S81D (3D mutant) with intact and thermally denatured actin was analyzed by means of fluorescence spectroscopy and ultracentrifugation. Wild type HSP25 did not affect the polymerization of intact actin. The HSP25 3D mutant decreased the initial rate without affecting the maximal extent of intact actin polymerization. G-actin heated at 40-45 degrees C was partially denatured, but retained its ability to polymerize. The wild type HSP25 did not affect polymerization of this partially denatured actin. The 3D mutant of HSP25 increased the initial rate of polymerization of partially denatured actin. Heating at more than 55 degrees C induced complete denaturation of G-actin. Completely denatured G-actin cannot polymerize, but it aggregates at increased ionic strength. HSP25 and especially its 2D and 3D mutants effectively prevent salt-induced aggregation of completely denatured actin. It is concluded that the interaction of HSP25 with actin depends on the state of both actin and HSP25. HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates.     

Changes in conformation and nucleotide binding of Ca, Mn, or MgG-actin upon removal of the bound divalent cation. Studies of ultraviolet difference spectra and optical rotation

The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca²⁺, Mn²⁺, or Mg²⁺ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn²⁺ > Ca²⁺ > Mg²⁺. ATP release is partially reversible upon Ca²⁺ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca²⁺, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca²⁺, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of staphylococcal nuclease denaturation. I. The acid-denatured state.

Using high-sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower delta Cp and delta H for the denaturation reaction. The acid-denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid-denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of delta H on pH.     

Instability of F-actin under the influence of β-actinin

When G-actin was polymerized in the presence of β-actinin, the resultant F-actin became unstable on incubation in 0.1 M KCl at 25°C. The F-actin filaments changed into globular aggregates of 20–30 nm in diameter. ATP protected the denaturation of β-actinin-modified F-actin. Thus the instability of the isolated I filaments is explained by the influence of β-actinin.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.     

Microcalorimetric study of the heat denaturation of carboanhydrase B

The microcalorimetric study of heat denaturation of carbonic anhydrase B has revealed that the process of denaturation of carbonic anhydrase B is accompanied by the formation of intermolecular complexes which are disrupted at a further increase of temperature. It is shown that zinc atoms stabilize the native state and do not influence the stability of intermolecular complexes.     

The unusually stable coiled-coil domain of COMP exhibits cold and heat denaturation in 4-6 M guanidinium chloride

A high thermal stability is observed for the five-stranded alpha-helical coiled-coil domain of cartilage oligomeric matrix protein COMP. It does not unfold in non-denaturing buffer between 0 and 100 degrees C and thermal denaturation is only achieved at high concentrations of guanidinium chloride (4-6 M). In these solutions the protein structure is lost at decreasing (cold denaturation) and increasing temperatures (heat denaturation). In the cold denaturation region, the melting profile showed deviations from the theory of Privalov et al. [P.L. Privalov, V. Griko Yu, S. Venyaminov, V.P. Kutyshenko, Cold denaturation of myoglobin, J. Mol. Biol. 190 (1986) 487-498] probably due to deviations from a two-state mechanism. High thermal stability as well as cold and heat denaturation was also observed for a mutant of the coiled-coil domain of COMP in which glutamine 54 was replaced by isoleucine but it still forms pentamer. The melting temperatures in plain buffer for the heat denaturation of COMP coiled-coil domain and its mutant obtained by extrapolation to zero molar guanidinium chloride concentration are approximately 160 and 220 degrees C, respectively, which groups them among the most stable proteins.     

19F nuclear magnetic resonance studies of selectively fluorinated derivatives of G- and F-actin

G-Actin is a globular protein (Mr 42 300) known to have three cysteine residues that are at least partially exposed and chemically reactive (Cys-10, -284, and -374). When G-actin was reacted with 3-bromo-1,1,1-trifluoropropanone, three resolvable 19F resonances were observed in the 19F NMR spectrum. This fluorinated G-actin derivative remained fully polymerizable, and its 31P NMR spectrum was not significantly different from that of unmodified G-actin, indicating that the chemical modification did not denature the actin and the modified residues do not interfere with the extent of polymerization or the binding of adenosine 5'-triphosphate. One of the three 19F resonances was assigned to fluorinated Cys-374 on the basis of its selective reaction with N-ethylmaleimide. This resonance was dramatically broadened after polymerization of fluorinated G-actin, while the other two resonances were not markedly broadened or shifted. Thus, Cys-10 and -284 are not involved in or appreciably affected by the polymerization of G-actin, while the mobility of the 19F label at Cys-374 is markedly reduced.     

Tryptophan phosphorescence of nascent and inactivated actin at the room temperature]

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.     

Quantitative analysis of G-actin transport in motile cells

Cell migration is based on an actin treadmill, which in turn depends on recycling of G-actin across the cell, from the rear where F-actin disassembles, to the front, where F-actin polymerizes. To analyze the rates of the actin transport, we used the Virtual Cell software to solve the diffusion-drift-reaction equations for the G-actin concentration in a realistic three-dimensional geometry of the motile cell. Numerical solutions demonstrate that F-actin disassembly at the cell rear and assembly at the front, along with diffusion, establish a G-actin gradient that transports G-actin forward “globally” across the lamellipod. Alternatively, if the F-actin assembly and disassembly are distributed throughout the lamellipod, F-/G-actin turnover is local, and diffusion plays little role. Chemical reactions and/or convective flow of cytoplasm of plausible magnitude affect the transport very little. Spatial distribution of G-actin is smooth and not sensitive to F-actin density fluctuations. Finally, we conclude that the cell body volume slows characteristic diffusion-related relaxation time in motile cell from ∼10 to ∼100 s. We discuss biological implications of the local and global regimes of the G-actin transport.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Comparative Study of Thermostability and Structure of Close Homologues - Barnase and Binase

Abstract

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2–6 have been determined. The barnase heat denaturation (pH 2.85.5) proceeds according to the “all-or-none” principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 °C at pH 5 to 7 °C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5–5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.     

A comparison of methods for sample clean-up prior to quantification of metal-binding proteins.

1. The recovery of rabbit metallothionein and metallothionein-like proteins has been examined under different conditions. 2. After heat or ethanol denaturation, recovery of rabbit MT-II was quantitative. 3. Recovery of rabbit MT was not affected by the presence or absence of dithiothreitol (DTT) after heat treatment. However, recovery from ethanolic solution decreased in buffers that did not contain the antioxidant. 4. Acetone precipitation of rabbit MT resulted in lower yields which approached 90% only in the presence of DTT. 5. Recovery of metallothionein-like proteins from cytosols of the hepatopancreas of the tanner crab, Chionoecetes bairdi, were examined using pulse polarography and HPLC. Relative to heat denaturation, ethanol and acetone yielded recoveries of 66 and 28%, respectively, in the presence of DTT. However, acetone did yield a solution which contained less extraneous protein of molecular mass greater than 43 kDa than heat denaturation. 6. We concluded that heat denaturation is the preferred treatment for quantitative recovery of metal-binding proteins. Acetone precipitation is useful for the purification of MT.     

Vertebrate slow skeletal muscle actin - conservation, distribution and conformational flexibility

The existence of a unique sarcomeric actin is demonstrated in teleosts that possess substantial amounts of slow skeletal muscle in the trunk. The slow skeletal isotype is conserved. There is one amino acid substitution between Atlantic herring slow skeletal actin and the equivalent in salmonids. Conversely, the intra-species variation is considerable; 13 substitutions between different herring skeletal isotypes (slow versus fast). The isomorphisms (non-conservative underlined: residues, 2, 3, 103, 155, 160, 165, 278, 281, 310, 329, 358, 360 and 363) are restricted to sub-domains 1 and 3 and include the substitution Asp-360 in 'slow' to Gln in 'fast' which results in an electrophoretic shift at alkaline pH. The musculature of the trunk facilitates the preparation of isoactins for biochemical study. Herring slow skeletal G-actin (Ca.ATP) is more susceptible to thermal, and urea, -induced denaturation and subtilisin cleavage than that in fast skeletal, but more stable than the counterpart in salmonids (one substitution, Gln354Ala) highlighting the critical nature of actin's carboxyl-terminal insert. Fluorescent spectra of G-actin isoforms containing the isomorphism Ser155Ala in complexation with 2'-deoxy 3' O-(N'-Methylanthraniloyl) ATP infer similar polarity of the nucleotide binding cleft. An electrophoretic survey detected two skeletal actins in some (smelt and mackerel) but not all teleosts. One skeletal muscle actin was detected in frog and bird.     

A comparative thermodynamic study of heat and cold denaturation of beta-lactoglobulin]

The changes in structure and thermodynamic parameters of beta-lactoglobulin upon heat and cold denaturation have been studied using both scanning microcalorimetry and circular dichroism spectroscopy methods. It has been shown that in contrast to the heat denaturation process, the cold denaturation of beta-lactoglobulin is accompanied by an opposite heat effect. In all cases, the calorimetrically measured enthalpy of beta-lactoglobulin cold denaturation is higher than it was expected from the two-state model of denaturation transition. It has been concluded that beta-lactoglobulin cold denaturation cannot be represented by a transition between two microscopic states--native and denatured. The latter, is due to the additional process that occurs together with the disruption of the beta-lactoglobulin tertiary structure and is accompanied by increasing heat capacity. Taking into account the heat capacity contribution of this process upon calculation of the enthalpy makes it closer to the enthalpy value calculated for the two-state model of denaturation transition.     

Association kinetics and binding constants of nucleoside triphosphates with G-actin.

The dissociation of the complex between 1:N6-ethenoadenosine, 5'-triphosphate (xiATP) and G-actin was initiated by dilution to concentrations between 1 micronM and 5 nM and monitored by the fluorescence change of xiATP. The results were quantitatively explained by a two-step mechanism: a reversible dissociation of the actin-nucleotide complex followed by a fast irreversible inactivation of nucleotide-free G-actin. Under normal conditions (0.8 mM CaCl2, pH 8.2,21 degrees C), the rate-limiting step was the dissociation of the nucleotide-G-actin complex. The half-time of the dissociation of xiATP from G-actin was 290 s as compared to only 13 s for the following denaturation step of nucleotide-free actin. 1 mM EDTA highly accelerated the dissociation step and, regardless of its concentration, the complex dissociated quantitatively within 1 min. Addition of Ca2+ within 20 s after EDTA addition induced a re-association of xiATP with nucleotide-free but still native G-actin. This reversal was kinetically resolved by means of a multimixing stopped-flow apparatus. The association rate constant was 6 X 10(6) M-1s-1. From the association and dissociation rate constant, a value of 2.5 X (10(9) M-1 was calculated for the binding constant of xiATP to G-actin. The binding constant of ATP (1.4 X 10(10) M-1) was derived from the relative binding constant of xiATP and ATP as determined by fluorescence titration of xiATP-G-actin with ATP. These binding constants are 10(3)-10(4) times higher than values reported earlier on the basis of more indirect data.