共查询到20条相似文献,搜索用时 15 毫秒
1.
N Go 《International journal of peptide and protein research》1975,7(4):313-323
A theoretical method is developed by which the character of the process of protein denaturation (e.g., whether or not it is of the all-or-none type) can be discussed in terms of conformation of native proteins and the forces stabilizing it. An important role is played by a quantity S(H): entropy of a protein molecule in solution in the conformational states with a given value of enthalpy H. It is demonstrated that the all-or-none type denaturation of proteins is a rather direct consequence of the globularity and specificity of the native conformations. Denaturations with significant intermediate states are discussed. Denaturations induced by added denaturants are also discussed. 相似文献
2.
The statistical thermodynamic model of protein structure proposed in paper I is developed with special attention to the hydrophobic interaction. Calorimetric measurements of the thermal denaturation of five globular proteins, ribonuclease A, lysozyme, alpha-chymotrypsin, cytochrome c, and myoglobin, are quantitatively analyzed using the model. The thermodynamic parameters obtained by the least squares method reflect the global, average properties of proteins and are in good agreement with the expected values estimated from experimental and theoretical studies for model peptides. The average bond energy epsilon is well related to the tertiary structure of each protein. However, the difference in the parameters between different proteins is not observed for the cooperative energy ZJ and the chain entropy alpha. The individuality of a protein as far as its structural stability is concerned, is mainly reflected by the parameter gamma specifying the hydrophobic nature of a protein. The model is further applied in the analysis of several aspects of the structural stability of globular proteins. Denaturation induced by denaturants, salts, and pH are also explained by the model in a unified manner. 相似文献
3.
Ragone R 《Proteins》2004,54(2):323-332
This article shows that the stability profiles of thermophilic proteins are significantly displaced toward higher temperatures as compared to those of mesophilic proteins. A similar trend characterizes the aqueous transfer of N-alkyl amides. In fact, as a general feature of transfer processes, liquid dissolution profiles are centered at temperatures higher than those of solid ones. This behavior is governed by packing contributions. A partition of the unfolding thermodynamics based on the analysis of phenomenological temperatures common to dissolution and unfolding phenomena provides a clue to understanding the mechanism of thermal stabilization. In fact, the position of stability profiles along the temperature axis does not appear to depend on solvation of internal residues. Instead, it is notably affected by solidlike components, whose progressive decrease appears to drive the heat denaturation temperature increase of most thermostable proteins. As a corollary, it is shown that there are actually two limiting mechanisms of thermal stabilization. 相似文献
4.
We previously reported a combinatorial strategy for designing alpha-helical proteins by assigning only the binary patterning of polar or nonpolar residues [Kamtekar, S., Schiffer, J. M., Xiong, H. Y., Babik, J. M., and Hecht, M. H. (1993) Science 262, 1680-1685]. Here we describe the finding that approximately half of the proteins in the original collection display some level of cooperativity in their thermal denaturation profiles. Many are monomeric in solution, demonstrating that the observed cooperativity is not merely a consequence of oligomerization. These findings demonstrate that although the combinatorial nature of the design strategy precludes explicit design of side-chain packing, binary patterning incorporates sufficient sequence information to generate de novo proteins with cooperatively folded structures. As binary partitioning of polar and nonpolar amino acids is an intrinsic part of the genetic code, these findings may bear on the early evolution of native proteins. 相似文献
5.
Cold denaturation of proteins 总被引:29,自引:0,他引:29
P L Privalov 《Critical reviews in biochemistry and molecular biology》1990,25(4):281-305
This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly temperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reevaluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive contributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed. 相似文献
6.
The advantageous usage of the high pressure technique in studies of cold denaturation of proteins is reviewed, with a brief explanation of the theoretical background of this universal phenomenon. Various experimental results are presented and discussed, explaining the plausible image of the cold denatured state of proteins. In order to understand more clearly this phenomenon and protein structure transition in general, several studies on model polymer systems are also reviewed. 相似文献
7.
Study of thermal properties and heat-induced denaturation and aggregation of soy proteins by modulated differential scanning calorimetry 总被引:2,自引:0,他引:2
The thermal properties and heat-induced denaturation and aggregation of soy protein isolates (SPI) were studied using modulated differential scanning calorimetry (MDSC). Reversible and non-reversible heat flow signals were separated from the total heat flow signals in the thermograms. In the non-reversible profiles, two major endothermic peaks (at around 100 and 220 degrees C, respectively) associated with the loss of residual water were identified. In the reversible profiles, an exothermic peak associated with thermal aggregation was observed. Soy proteins denatured to various extents by heat treatments showed different non-reversible and reversible heat flow patterns, especially the exothermic peak. The endothermic or exothermic transition characteristics in both non-reversible and reversible signals were affected by the thermal history of the samples. The enthalpy change of the exothermic (aggregation) peak increased almost linearly with increase in relative humidity (RH) in the range between 8 and 85%. In contrast, the onset temperature of the exotherm decreased progressively with increase in RH. These results suggest that the MDSC technique could be used to study thermal properties and heat-induced denaturation/aggregation of soy proteins at low moisture contents. Associated functional properties such as water holding and hydration property can also be evaluated. 相似文献
8.
Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. 总被引:13,自引:7,他引:6
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed. 相似文献
9.
Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed. 相似文献
10.
Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat-shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat-shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form. 相似文献
11.
Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation. 总被引:12,自引:9,他引:3
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
P. D. Thomas K. A. Dill 《Protein science : a publication of the Protein Society》1993,2(12):2050-2065
How important are helical propensities in determining the conformations of globular proteins? Using the two-dimensional lattice model and two monomer types, H (hydrophobic) and P (polar), we explore both nonlocal interactions, through an HH contact energy, as developed in earlier work, and local interactions, through a helix energy, σ. By computer enumeration, the partition functions for short chains are obtained without approximation for the full range of both types of energy. When nonlocal interactions dominate, some sequences undergo coil-globule collapse to a unique native structure. When local interactions dominate, all sequences undergo helix–coil transitions. For two different conformational properties, the closest correspondence between the lattice model and proteins in the Protein Data Bank is obtained if the model local interactions are made small compared to the HH contact interaction, suggesting that helical propensities may be only weak determinants of globular protein structures in water. For some HP sequences, varying σ/ leads to additional sharp transitions (sometimes several) and to “conformational switching” between unique conformations. This behavior resembles the transitions of globular proteins in water to helical states in alcohols. In particular, comparison with experiments shows that whereas urea as a denaturant is best modeled as weakening both local and nonlocal interactions, trifluoroethanol is best modeled as mainly weakening HH interactions and slightly enhancing local helical interactions. 相似文献
12.
An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods. 相似文献
13.
Chromatin was prepared from the buds and cotyledons of Alaskapea seedlings. The dissociated chromosomal components in thepresence of 2 M NaCl and 5 M urea were completely fractionatedinto DNA and proteins with a Bio-Gel A50 column. The proteinswere recovered by (NH4)2SO4 and further fractionated into histonesand non-histone proteins using a Bio-Rex 70(Na+) column. Thedifference in the ratios of histones to non-histone proteinsbefore and after chromatography with the Bio-Rex 70 was lessthan 10%. The histones and non-histone proteins thus preparedshowed typical protein absorption spectra. Polyacrylamide gelelectrophoresis of histones showed that the histone compositionsin buds and cotyledon were similar, but the amount of HI histoneswas a little less in cotyledons than in buds. Unlike histones,non-histone proteins fractionated by SDS-polyacrylamide gelelectrophoresis indicated distinct differences between the twotissues. Buds had more heterogeneous non-histone proteins, atleast 13 polypeptides, than cotyledons did. On the other hand,non-histone proteins of cotyledons showed less heterogeneityand lacked proteins of high molecular weight which were foundin buds. (Received May 6, 1976; ) 相似文献
14.
The three states of globular proteins: acid denaturation. 总被引:2,自引:0,他引:2
We describe statistical mechanical theory that aims to predict protein stabilities as a function of temperature, pH, and salt concentration, from the physical properties of the constituent amino acids: (1) the number of nonpolar groups, (2) the chain length, (3) the temperature-dependent free energy of transfer, (4) the pKa's (including those in the native state) and their temperature dependencies. We calculate here the phase diagrams for apomyoglobin and hypothetical variant proteins. The theory captures essential features of protein stability including myoglobin's Tm vs pH as measured by P. L. Privalov [(1979) Advances in Protein Chemistry, Vol. 33, pp. 167-241] and its ionic strength vs pH phase diagram as measured by Y. Goto and A. L. Fink [(1990) Journal of Molecular Biology, Vol. 214, pp. 803-805]. The main predictions here are the following: (1) There are three stable states, corresponding to native (N), compact denatured (C), and highly unfolded (U), with transitions between them. (2) In agreement with experiments, the compact denatured state is predicted to have enthalpy closer to U than N because even though there is considerable hydrophobic "clustering" in C, this nevertheless represents a major loss of hydrophobic contacts relative to configurations (N) that have a hydrophobic "core." (3) C becomes more prominent in the phase diagram with increasing nonpolar content or decreasing chain length, perhaps thus accounting for (a) why lysozyme and alpha-lactalbumin differ in their denatured states, and (b) why shortened Staph nuclease molecules are compact. (4) Of major importance for protein calorimetry is Privalov's observation that the enthalpy of folding, delta H (T, pH) is independent of pH. The theory accounts for this through the prediction that the main electrostatic contribution to stability is not enthalpic; the main contribution is the entropy, mainly due to the different distributions of protons and small ions in the native and denatured states. 相似文献
15.
The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C. 相似文献
16.
The proteins of adults worms (male and female) of two isolates (BH and RJ) of Schistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of male and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females proteins were detected by Western blotting using a sera from infected Nectomys squamipes. 相似文献
17.
High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10?3M Na+. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle. 相似文献
18.
Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli. 总被引:5,自引:3,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes. 相似文献
19.
Solvent denaturation and stabilization of globular proteins 总被引:17,自引:0,他引:17
Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation. 相似文献
20.
L Cocco A Facchini N M Maraldi S Capitani F A Manzoli 《Physiological chemistry and physics》1976,8(4):319-325
Histones and non-histone chromosomal proteins were characterized in B and T human lymphocytes by means of polyacrylamide disc gel electrophoresis. It was found that while histones do not present appreciable differences in the two examined populations, non-histone chromosomal proteins exhibit distinct electrophoretic profiles. Low molecular weight proteins predominate in B lymphocytes whereas high and intermediate proteins are largely represented in T lymphocytes. The latter proteins may be related to the capability of these resting cells to proliferate under appropriate antigenic stimuli. 相似文献