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PubMed1977

Structural changes and fluctuations of proteins. II. Analysis of the denaturation of globular proteins.

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.

Calorimetry, Differential Scanning↗
PubMed1978

Renaturation of a reduced Taiwan cobra cardiotoxin.

Refolding of a denatured protein obtained by reducing cardiotoxin from the Taiwan cobra with mercaptoethanol has been carried out in aqueous and non-aqueous solutions. Oxidation of the reduced protein in 0.05 M phosphate buffer (pH 7.2) resulted in isolating an active protein which showed, as compared to native cardiotoxin, identical conformation and biological activities such as lethality, antigenicity and muscle contracture inducing activity. On the other hand, the reduced protein was undergoing incorrect SS-pairing and several inactive products were formed in a mixture of 1,2-ethanediol and 1-propanol (1 : 1; v/v).

Animals↗
PubMed1979

Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.

A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.

Animals↗
PubMed1975

Digestion of insulin derivatives with subtilisin: a kinetic study.

Native, denatured, performic acid-oxidized or S-sulfo insulin and S-sulfo or performic acid-oxidized A- and B-chains were digested with subtilisin type Carsberg. The proteolysis was followed by measuring the uptake of alkali through autotitration. The kinetic study shows the existence of 2 first-order reaction classes which differ markedly in rate constant. The number of bonds split with fast and with slow reactions has been calculated. Only one of a total of 12 cleavable bonds in native insulin is opened by fast reaction. In the denatured protein the number of bonds split by the fast reaction increases to 4 and in the oxidized and S-sulfo protein 3 bonds are cleaved, while the slow cleavable bonds number 2 and 7, respectively, The kinetic study of the proteolysis of S-sulfo A-chain and of oxidized or S-sulfo B-chain shows that two bonds are split in A-chain with the fast and slow reactions, while in B-chain only one of the six cleavable bonds is susceptible to fast attack.

Hydrogen Bonding↗
PubMed1978

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.

Amino Acids↗
PubMed1979

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.

Animals↗
PubMed1978

Myxobacterial slime and proteolytic activity.

An extracellular protein-polysaccharide-lipide (PPL) complex from exponentially growing cultures of Myxococcus virescens was purified by phosphate precipitation and gel chromatography. The high molecular weight slime polymer appeared homogenous upon isoelectric focusing. The PPL complex exhibited proteolytic activity against gelatin and the activity was only partly reduced by heat treatment. The function of the slime polymer as protein denatured was studied. The complex formed micelles similar to anionic detergents and it inhibited the precipitation and coagulation of proteins by trichloroacetic acid. Lysozyme was totally inactivated when treated with the PPL complex. By gel chromatography binding studies, the PPL complex was found to bind lysozyme in the ratio of 1 to 5.8 (w/w). After separation of added protein from the complex the anticoagulation effect on the protein remained. The biological function of the PPL complex was demonstrated with hemoglobin. When all susceptible peptide bonds in PPL-treated hemoglobin were hydrolyzed by trypsin only 20% in the urea-denatured protein were attacked. The combined role of slime and proteolytic activity is discussed.

Bacterial Proteins↗
PubMed1978

Denaturation of proteins by ascorbic acid: effects on dopamine-beta-hydroxylase.

Under conditions that are optimum for DbetaH, ascorbic acid denatures serum albumin, gamma-globulin, catalase, and DbetaH. With ascrobate plus Cu2+, the proteins are almost completely destroyed. Pyrazole protects DbetaH and albumin, but not catalase. Superoxide dismutase (SOD) is not denatured by ascorbate, with or without Cu2+, and in combination with catalytic amounts of catalase or Fe2+ it stimulates maximum DbetaH activity. In other words, a combination of catalase and SOD, or Fe2+ and SOD, will protect DbetaH. Excessive amounts of catalase and/or other protein, either native or denatured will prevent the effects of superoxide and/or ascrobate, but cannot replace the requirements for catalytic quantities of catalase or Fe2+. The results suggest that the rate of hydroxylation of tyramine may be limited by superoxide, but that the latter per se does not denature DbetaH as does hydrogen peroxide. The in vitro activation of oxygen by DbetaH is a toxic process, involving the production of both hydrogen peroxide and superoxide and possibly other free radicals. In the absence of precise regulation of the production and concentrations of these compounds, the enzyme is denatured.

Animals↗
PubMedDate not supplied

Thermodynamics of aging in Drosophila melanogaster.

The data on mortality kinetics and decline in functions reported in the preceding article are used to calculate temperature coefficients for the aging process(es) in Drosophila. Different values are found, according to the model chosen to account for the mortality kinetics. The respective implications of three equally suitable models are discussed. Thus, organization parameters on two different levels can be identified: rates of changes assumed to occur at the elementary molecular level, and redundancy factors at a more integrated level. Their temperature coefficients are compared with those of protein denaturations and lipid peroxidation pigment accumulation. It is suggested that elementary molecular processes responsible for aging can indeed be protein denaturations, whereas the known lipid peroxidation pigment accumulation is more likely to be a secondary effect, resulting from a failure of the overall cellular organization at a more integrated, supramolecular, level.

Aging↗
PubMed1976

Statistical mechanics of protein folding, unfolding and fluctuation.

Conformational fluctuations of globular proteins in the native state and the processes of folding and unfolding are studied from the statistical mechanical point of view. 1) It is pointed out that the formation of the native specific conformation of a globular protein is not a result of random sampling of minimum energy conformations. This fact provides a motivation for the study of the processes of folding, or the paths of folding, of proteins. The processes of folding and unfolding are shown to be statistical mechanical in nature. 2) The conformational (folding and unfolding) transitions in globular proteins are compared with the helix-coil transitions in polypeptides. The gradedness of the latter transition is shown to be due to the fact that the phenomenon is of an essentially one-dimensional system. The former transition is characterized ideally by the fact that it is of the all-or-none type. 3) A lattice model of proteins is introduced. "A protein molecule" is defined as a chain of noninteresting units of a given length on a two-dimensional square lattice. The copolymeric character of protein molecules is incorporated into the model by specificities of interunit interactions. 4) This model proved powerful for studying the statistical mechanical characterization of protein denaturation and fluctuations. The specificities of interunit interactions were shown to be the primary factors responsible for the all-or-none type transition from native to denatured states of globular proteins. 5) The model is studied by the Monte Carlo method of Metropolis et al., which simulates a kinetic process approximately. The method is shown to be a promising tool in finding the native conformation of proteins from their amino acid sequence. 6) A new theoretical method is developed to study phenomenologically the processes of protein folding and unfolding and the conformational fluctuations in the native state. An important role is played by a quantity S(H): the entropy of a protein molecule in solution in the conformational states with a given value of enthalpy H. Qualitative character of the S-H curve, such as whether it is convex or concave determines characteristics of the conformational transition in a globular protein such as whether or not it is of the all-or-none type. 7) The concept of an ideal process of protein folding and unfolding is introduced and defined by three statements. The S-H curve is calculated for this ideal process. The curve is shown to be concave, indicating that the transition is of the all-or-none type. This conclusion is drawn essentially from the globularity and specificity of the native conformational of proteins. 8) Residual structures in the denatured state and conformational fluctuations in the native state are discussed. In order to discuss the latter, an independent fluctuating site model is introduced, in which it is assumed that there are several independent fluctuating sites, each localized in some part of the protein molecule...

Computers↗
PubMed1976

Effects of the aliphatic carboxylate series of salts on the conformation of proteins.

The effects of the aliphatic acid series of salts, formate, acetate, propionate, butyrate, valarate, and caproate, on the conformation of sperm whale myoglobin, human hemoglobin A, and horse heart cytochrome c were investigated by spectral measurements in the Soret region, optical rotation, and intrinsic viscosity measurements. The effectiveness of the aliphatic acid salts as unfolding reagents for proteins is found to increase with increasing hydrocarbon content of the alkyl chains of the salts, which is analogous in behavior to effects of the urea, amide, and alcohol series of protein denaturants. The denaturation midpoints, Sm, as a function of the unfolding reagent were analyzed using the equations of Peller (Peller, L. (1959), J. Phys, Chem- 63, 1199) and Flory (Flory, P.J. (1957), J. Cell. Comp. Physiol. 49, 175) with binding constants based in part on the Scherage-Nemethy theory of hydrophobic bonding or evaluated from free-energy transfer data of nonpolar amino acid side chains from aqueous to nonaqueous solvents. The summation of the polar KP and hydrophobic KHphi contributions of solvent to protein amino acid side chain interactions were found to give best account of the protein denaturation data. Intrinsic viscosity and optical rotation data obtained on hemoglobin and myoglobin at high salt concentrations, above the unfolding transition regions, indicate that the product of denaturation by the aliphatic acid salts is less unfolded than in 6 M guanidine hydrochloride solutions. Residual elements of the helical regions of the proteins seem to either escape unfolding or are reformed at high concentrations of the denaturing salts.

Binding Sites↗
PubMed1976

Injury of the cell's respiratory system by heat and by formaldehyde. Thermokinetics and early molecular events.

This is a study of the manner in which the respiratory system of the cell is injured either by elevated temperature or by exposure to diluted formaldehyde. Molecular mechanisms were identified by thermokinetic measurements. The rates at which respiratory failure developed in mouse liver slices in an injurious environment were measured at various temperatures. The data were fitted to the Arrhenius equation, and the effective activation energies of the injury processes were calculated. These data show that (1) the thermokinetics of injury to the cell's respiratory system, whether by thermal or chemical means, follows the Arrhenius law. (2) Thermal injury of the cell's respiratory system has a high activation energy, indicating that the critical, rate-determining event is a protein denaturation. Other mechanisms such as imbalance of metabolic reaction rates and thermal liquefaction of membrane lipids can be ruled out. (3) Repression of cell respiration by diluted formaldehyde has an activation energy compatible with a chemical reaction but low enough to exclude protein denaturation as a mechanism.

Animals↗
PubMed1978

Resistance of lipophilin, a hydrophobic myelin protein, to denaturation by urea and guanidinium salts.

The influence of urea, guanidinium chloride (GdmCl), and granidinium thiocyanate (GdmSCN) on the solution structure of lipophilin was examined by circular dischroism and fluorescence techniques. According to the CD results, lipophilin retained at least 60% of organized secondary structure in 8 M urea and 6 M GdmCl (measurements were not possible in GdmSCN). This partial denaturation was of a complex, irreversible nature, and was not appreciably enhanced by prolonged incubation (8 days), by heating to 70 degrees C, by disulfide bond reduction, or by pH variation in the range pH 1.5 to 11. Fluorescence studies demonstrated that the tryptophan residues were only slightly perturbed by 8 M and 6 M GdmCl and remained well buried to the permeant quenching agent acrylamide. A greater, but still far from complete, disruption of lipophilin was achieved in 6 M GdmSCN, and fluorescence polarization provided evidence for some form of cooperative structural change induced by increasing concentrations of this reagent. Transfer of the protein from 2-chloroethanol, in which the tryptophan residues are fully exposed, into 6 M GdmSCN by dialysis resulted in reburial of the fluorophores owing to development of tertiary structure. The combined evidence suggests that the extraordinary resistance of lipophilin to these denaturants is due to the presence of an impervious hydrophobic core. In lipophilin and some other membrane-associated proteins, extended sequences of apolar residues might provide the nuclei for such structural domains.

Circular Dichroism↗