Microwave dielectric study on bound water of globule proteins in aqueous solution

A dielectric relaxation peak due to bound water of globule proteins in aqueous solution was observed at first by the use of a time domain reflectometry. This peak locates around 100 MHz as well as that of the aqueous DNA solution and the moist collagen, and has a relaxation strength in proportion to surface of the globule protein except for trypsin and pepsin of hydrolase. It is suggested that this peak is caused by orientation of bound water molecules on the protein surface. The number of bound water molecules estimated is in good agreement with that obtained by other method such as x-ray analysis. The solution exhibits another peak below 100 MHz, which is caused by the rotation of globule protein supplemented by migration of the counterion. Its relaxation time is completely proportional to the molecular weight of the protein. © 1994 John Wiley & Sons, Inc.     

Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels

Outer membrane protein of Escherichia coli prepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.     

Microwave dielectric study on hydration of moist collagen

Two dielectric relaxation peaks were found in moist collagen by the time domain reflectometry. The low-frequency peak around 100 MHz moves little as the water content is varied. Its relaxation strength depends on the content and vanishes for completely dried collagen. This process is concluded to be due to water molecules strongly bound to the tropocollagen. Amount of the bound water is estimated as 0.12 g water/g collagen. Twenty-one water molecules are bound to one repeat of the triple helix. The existence of stringlike water chains is suggested. If the water content is less than 0.5 g water/g collagen, the high frequency peak locates between those of bound and bulk water. Water among the tropo-collagen is weakly bound to the collagen. In the higher region it does not change much with the content, being close to that of bulk water. The bulk water appears in this region.     

Structure of bovine pancreatic phospholipase A2 at 1.7 Å resolution

The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å (Dijkstra et al., 1978). This model was adjusted to the multiple isomorphous replacement map with Diamond's real space refinement program (Diamond, 1971,1974) and subsequently refined using Agarwal's least-squares method (Agarwal, 1978). The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH₊³ group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.     

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.     

Dielectric study of the cooperative order–disorder transition in aqueous solutions of schizophyllan,a triple-helical polysaccharide

Schizophyllan exists in aqueous solution as a triple helix, which is intact at room temperature. Its aqueous solution forms some ordered structure at low temperatures but undergoes a sharp transition to a disordered structure as the temperature is raised. The transition temperature T_c is about 7 and 18°C for H₂O and D₂O solutions, respectively. This transition was followed by time-domain reflectometry to investigate dynamic aspects of the transition. In addition to a major peak around 10 GHz, the dielectric dispersion curve of a 20 wt % schizophyllan in D₂O exhibited a small peak around 100 MHz below T_c and around 10 MHz above T_c. The major peak is due to bulk water, whereas the 100 MH_z peak is assigned to “bound” or “structured” water, and that around 10 MHz to side-chain glucose residues. However, unlike usual bound water reported for biopolymer solutions, this “structured” water disappears abruptly when the temperature becomes close to T_c without accompanying a conformational transition of the main chain. The above assignment is consistent with the structure of the ordered phase derived from previous static data that it consists of side-chain glucose residues along with nearby water molecules surrounding the helix core that are interacting with each other loosely through hydrogen bonds, and spreads radially only a layer of one or two water molecules but a long distance along the helix axis. © 1995 John Wiley & Sons, Inc.     

Thermogravimetric analysis of human femur bone

Thermal decomposition analysis of human femur bone has been carried out in the temperature range between 25°C and about 1000°C. In this temperature range, the differential primary weight loss curve yielded four distinct peaks for the femur bone. Each minimum inflexion point was arbitrarily chosen as the temperature at which the thermal decomposition of the component responsible for the peak had been completed. Aided by the thermogravimetric data on tendon collagen, the observed four peaks have been identified as follows: First: dissociation of water from the collagen; second: decomposition of collagen molecule itself; third: residual organic components associated with the collagen; fourth: water loss accompanied with the transformation of inorganic apatite and residual protein that were not decomposed at lower temperature. The fourth peak identified with the crystal transformation has been linked to the transformation ofα-tricalcium phosphate, which is known to have a hexagonal structure, toβ-tricalcium phosphate, which has a rhombohedral structure. This transformation has been observed between 700°C and 780°C which is in accord with the range observed in the transformation of synthetic apatite crystals. The experiments were performed under two different conditions: in normal pressure (in air) and in a reduced pressure of about 30μ Hg. In the average, the total weight loss up to the temperature of about 1000°C was about 35.64% for the heating in air and 36.45% for the heating in the reduced pressure. The weight loss has been carefully analyzed in the different temperature intervals and has been compared with an expected weight loss predicted by three most quoted formulas advanced for the apatite in bone. In addition to this, the conventional thermogravimetric analysis has been carried out in order to retrieve the thermal kinetic decomposition parameters corresponding to each peak. The activation energies corresponding to the first, second, and third peaks have been found to be slightly higher than those of tendon collagen. A detailed analysis showed that the bone contains about 28% protein and about 8% water (apatite crystalline water included).     

Index to Authors,Volume 7

Abstract

The thermally stimulated depolarization current (TSDC) measurements in frozen aqueous solutions, gels and solid layers of NaDNA show typically up to three dipolar overlapping peaks in the low-temperature range of 80—;150 K. Up to four discrete relaxation peaks have been observed at higher temperatures above 150 K. The low-temperature TSDC peaks are due to the dipolar relaxations of free and loosely bound water which crystallizes. Part of bound water especially in the first hydration shell of DNA molecule is at low temperatures in the form of glass. The transition of this glass from solidlike behavior to liquidlike behavior observed mainly in gels and solid samples is associated with a previously founded TSDC relaxation peak. The peak is at its maximum at 165- 250 K depending on the sample humidity. Existence of this relaxation in the samples with water contents in a broad range confirms, that the slowly relaxing shell (minimally 5–7 water molecules/nucleotide) closely associated with DNA double helix retains its characteristics. Also another peak of the high-temperature band at 180–205 K which was observed in the samples at hydration 2–1800 g H₂O/g dry NaDNA is due to a relaxation in the sample volume. At the highest temperatures relax the space charges trapped at the electrodes.     

Solubilization and partial purification of steroid sulfatase of human placenta

Steroid sulfatase of human placenta has been solubilized by treatment of the microsomal fraction with an amphoteric surface active agent, Miranol H2M and ultrasound. Criteria of solubility include non-sedimentation of the activity following centrifugation at 160,000 × g, its retention on Sepharose 6B and a single peak of activity after polyacrylamide gel electrophoresis. Enzyme activity was located in the same gel fractions for the two substrates tested; cholesterol sulfate and dehydroisoandrosterone sulfate. The addition of dithiothreitol was found necessary to maintain the stability of the enzyme indicating the presence of sulfhydryl groups in the molecule. A molecular weight of approximately 330,000 has been estimated from the elution volume of the enzyme system on a column of Sepharose 6B. It is believed that this protein represents a sulfatase enzyme complex composed of subunits with different specificities. From kinetic studies, a K_m of 6.2 × 10^?5M for the cleavage of dehydroisoandrosterone sulfate and a K_m of 2 × 10^?6M for the cleavage of cholesterol sulfate have been calculated.     

A molecular dynamics study of solvent behavior around a protein

The solvent structure and behavior around a protein were examined by analyzing a trajectory of molecular dynamics simulation of thetrp-holorepressor in a periodic box of water. The calculated selfdiffusion coefficient indicated that the solvent within 10 Å of the protein had lower mobility. Examination of the solvent diffusion around different atoms of different kinds of residues showed no general tendency. Thisfact suggested that the solvent mobility is not influenced significantly bythe kind of the atom or residue they solvated. Distribution analysis aroundthe protein revealed two peaks of water oxygen: a sharp one at 2.8 Å around polar and charged atoms and a broad one at ～3.4 Å aroundapolar atoms. The former was stabilized by water–protein hydrogen bonds, and the latter was stabilized by water-lwater hydrogen bonds, suggesting the existence of a hydrophobic shell. An analysis of protein atom–water radial distribution functions confirmed these shell structures around polar or charged atoms and apolar ones. © 1993 Wiley-Liss, Inc.     

Distributions of water around amino acid residues in proteins

The atomic co-ordinates from 16 high-resolution (less than or equal to 1.7 A = 0.1 nm), non-homologous proteins have been used to study the distributions of water molecule sites around the 20 different amino acid residues. The proportion of residues whose main-chain atoms are in contact with water molecules was fairly constant (between 40% and 60%), irrespective of the nature of the side-chain. However, the proportion of residues whose side-chain atoms were in contact with water molecules showed a clear (inverse) correlation with the hydrophobicity of the residue, being as low as 14% for leucine and isoleucine but greater than 80% for asparagine and arginine. Despite the problems in determining accurate water molecule sites from X-ray diffraction data and the complexity of the protein surface, distinct non-random distributions of water molecules were found. These hydration patterns are consistent with the expected stereochemistry of the potential hydrogen-bonding sites on the polar side-chains. The water molecules around apolar side-chains lie predominantly at van der Waals' contact distances, but most of these have a primary, shorter contact with a neighbouring polar atom. Further analysis of these distributions, combined with energy minimization techniques, should lead to improved modelling of protein structures, including their primary shells of hydration.     

用差示扫描量热计对低水含量蛋白质热变性前峰的研究

本文用P/EDSC-2型差示扫描量热计检测了11个具低水含量的高纯蛋白质的DSC曲线.发现它们无一例外地在变性峰前都具有小的吸热峰,即热变性前峰.实验表明变性峰和前峰的峰温、峰面积都强烈现依赖于蛋白质的含水量,而且样品被第一次扫描后在室温放置一定时间后前峰仍可以再现,再现前峰的峰温和峰面积取决于样品的水合度、第一次被扫描的终止温度以及第一次扫描后在室温放置的时间.本文还检测了一些多聚物、氨基酸、多肽以及热变性后蛋白质的DSC曲线.发现热变性后的蛋白质仍可出现前峰,但变性峰不再出现,显然两个峰的出现机制不同.本文并就前峰出现的可性能机制进行了初步讨论.     

Seasonal changes in the physical state of crown water associated with freezing tolerance in winter wheat

The relationship between freezing tolerance (expressed as LT₅₀, the lethal freezing temperature for 50% of plants) and the amount and physical state (as determined by spin-lattice [T₁] and spin-spin [T₂] relaxation times of protons) of water in crown tissue was examined in contrasting winter wheat (Triticum aestivum L.) varieties grown under field conditions from 1992 to 1994. During acclimation, the LT₅₀ values decreased from around -7 to -17, -20 and -27°C in PI 173438, Chihokukomugi and Valuevskaya, respectively. Tissue water content decreased continuously through autumn to reach a plateau around 3 g H₂O (g dry weight)^-1 in early winter when LT₅₀ was still decteasing, and then gradually increased under snow cover. A significant negative correlation was found between mean minimum air temperatures and freezing tolerance prior to the establishment of continuous snow cover. In contrast, a positive association between mean minimum temperatures and crown tissue water content was significant only when air temperatures were above 0°C, as water content did not decrease further at sub-zero temperatures. Seasonal changes in T₁ were closely related to changes in freezing tolerance. T₁ decreased until January even though water content stopped decreasing. Further tests on 15 field-grown varieties confirmed a strong negative association between freezing tolerance and T₁. The results suggest that cold hardening is comprised of two stages, with the transition occurring at ca 0°C. Development of hardiness was related to (1) a reduction in water content in the first stage (at minimum temperatures > 0°C), and (2) a change in physical state of water without much reduction in water content in the second stage. Varietal differences in hardiness thus arise due to changes in both water content and physical state of water.     

Circular Thin-layer Chromatography of Oligosaccharides

The physicochemical properties and chemical constituents of the blue protein from rice bran were investigated. The blue protein was a copper-containing glycoprotein, the molecular weight of which was found to be 18,300 Daltons by the sedimentation equilibrium method assuming the partial specific volume 0.72 cm³ g^?1. The hexose and pentose contents were 5.49 and 4.01 g per 100 g protein respectively. The copper content was 0.38% which corresponded to 1.09 atoms per one molecule of the protein. The electron spin resonance spectrum showed that the copper was in a cupric state. The standard oxidation-reduction potential of the copper was found to be +275 mV at 20°C and at pH 7.39. The visible and near infrared absorption maxima were found at 450, 600 and 890 mμ, and the 450 mμ band was optically active in the optical rotatory dispersion exhibiting a large Cotton effect.     

Active site dynamics of acyl-chymotrypsin

The motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl-chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH^? for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His-57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the N^ε2 of His-57 and is heldin place through a network of hydrogen-bonded water molecules in theactive site. The water molecule was found as close as 2.8 Å to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His-57-N^ε2 and Ser-195-0^γ with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His-57 imidazole ring between positions suitable for providing general-base catalyzed assistance and for providing general-acid catalyzed assistance may notbe required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His-57, Ser-195, and Asp-102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X-ray crystal-lographic data of γ-chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site. © Wiley-Liss, Inc.     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.     

The influence of dietary and waterborne zinc on heat-stable metal ligands in rainbow trout, Salmo gairdneri Richardson: quantification by 109Cd radioassay and evaluation of the assay

The ¹⁰⁹Cd binding assay of Eaton & Toal was critically evaluated and then used to assess the induction of cytosolic metal-binding ligands in rainbow trout exposed to Zn in the diet and/or in the water for 16 weeks. With purified rabbit Cd-Zn metallothionein (MT), ¹⁰⁹Cd binding and total Cd recovery in the assay were linear up to 5 μg of protein; gel chromatography revealed a single peak. With heat-denatured extracts of gill, liver and intestine from control and Cd- and Zn-injected trout, ¹⁰⁹Cd binding was generally linear with sample size. Gel chromatography demonstrated that ¹⁰⁹Cd was bound by a protein with the same apparent weight as MT (∼ 11000 daltons), but significant binding occurred also at three other regions [molecular weight (mol wt) >70 000, 30000 and <3000]. In the dietary/waterborne Zn exposure, induced ¹⁰⁹Cd-binding activity occurred not in the MT peak but in the low mol wt peak (< 3000). Activity in the gill rose in response to both dietary and waterborne Zn, but the liver did not respond. The maximum five-fold elevation in the gill was primarily a waterborne effect. In the intestine, the maximum rise was 25-fold due to both factors. The thresholds for induction were > 39 μg Zn^| in water, and > 90 mg kg ^| in the diet, but only when waterborne Zn was also high. There was no correlation between ¹⁰⁹Cd binding and acid soluble thiol levels, which tended to decline at higher Zn exposures.     

Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes

An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.     

Temperature-related heart rate in water and air and a comparison to other temperature-related measures of performance in the fiddler crab Leptuca pugilator (Bosc 1802)

Performance in poikilotherms is known to be sensitive to temperature, often with a low-sloping increase with temperature to a peak, and a steep decline with increasing temperature past the peak. We complemented past measures of performance by measuring heartbeat rates of the fiddler crab Leptuca pugilator in water and in air as a function of a range of temperatures previously shown to affect other measures of performance. In water over a range of 20–50 °C, heartbeat increased steadily to a peak at 40 °C and then steeply declined to near zero at 50 °C. In air, heartbeat also increased, but to a peak at 35 °C and then with a gentler decline than was found in water. Part of this different response may be due to evaporative water loss, which reduced body temperature in air, and therefore thermal stress, relative to body temperature when crabs were immersed in water. Increased availability of oxygen from air, according to the oxygen and capacity-limited thermal tolerance hypothesis, likely increased aerobic scope past the thermal peak, relative to within water, where oxygen delivery at higher temperatures may have been curtailed.We compared the heart rate performance relations to two previous measures of performance – endurance on a treadmill and sprint speed, both done in air. The peak performance temperature increased in the order: treadmill endurance time, sprint speed, heart rate in air, and heart rate in water, which demonstrates that different performance measures give different perspectives on the relation of thermal tolerance and fitness to temperature. Endurance may therefore be the limiting upper thermal stress factor in male fiddler crabs, when on hot sand flats. Temperature preference, found to be for temperatures <30 °C in air, could be a bet-hedging evolutionary strategy to avoid aerobic scope affecting endurance.     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)