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1.
Hemoglobin and the proteins of the crystalline lens contain active SH groups while in the native state, the number of active groups increasing as the pH rises. All the SH groups of denatured globin and of the denatured lens proteins are active at a pH so low that practically none of the SH groups of native hemoglobin and of native lens protein are active. The effect of denaturation on the SH groups of a protein is to extend towards the acid side the pH range of their activity. It is possible to oxidize the iron-porphyrin and the SH groups of hemoglobin independently of each other. 相似文献
2.
The two isofunctional enzymes aspartokinases-homoserine dehydrogenases I and II from Escherichia coli K 12 are compared using immunochemical techniques. The antibodies raised against one of these two proteins when in its native state can only recognize the homologous antigen, whether it is native or denatured. Contrarily, the antibodies raised against one of these two proteins when in its denatured state can recognize both the homologous and heterologous denatured antigens. The existence of this cross-reaction only between the two denatured aspartokinases-homoserine dehydrogenases suggests that these two enzymes have some similarity since such a reaction is not detected with several other denatured proteins. The regions involved in this similarity are buried inside the native proteins, and become exposed only upon denaturation. The same results, the existence of a cross-reaction between denatured species and none between the native ones, is obtained with proteolytic fragments derived from these two proteins and endowed with homoserine dehydrogenase activity. This resemblance between the two aspartokinases-homoserine dehydrogenases suggests that these proteins derive from a common ancestor. It is also proposed that such a cross-reaction between two denatured proteins is evidence for an homology between their amino acid sequences, and that the use of denatured proteins as both immunogens and antigens could be useful in detecting sequence homologies. 相似文献
3.
A. E. Mirsky 《The Journal of general physiology》1941,24(6):725-733
1. The same number of SH groups reduces ferricyanide in surface films of egg albumin as in albumin denatured by urea, guanidine hydrochloride, Duponol, or heat, provided the ferricyanide reacts with films while they still are at the surface and with the denatured proteins while the denaturing agent (urea, heat, etc.) is present. 2. The SH groups of a suspension of egg albumin made by clumping together many surface films react with ferricyanide in the same sluggish and incomplete manner as do the groups in egg albumin denatured by concentrated urea when the urea is diluted or in albumin denatured by heat when the solution is allowed to cool off. 3. The known change in configuration of the egg albumin molecule when it forms part of a surface film explains why SH groups in the film react with ferricyanide whereas those in native egg albumin do not. In the native egg albumin molecule groups in the interior are inaccessible to certain reagents. A film is so thin that there are no inaccessible groups. 4. Because of the marked resemblance in the properties of egg albumin in surface films and of egg albumin after denaturation by the recognized denaturing agents, it may be supposed that the same fundamental change takes place in denaturation as in film formation—indeed, that film formation is one of the numerous examples of denaturation. This would mean that in general the SH groups of denatured egg albumin reduce ferricyanide and react with certain other reagents because they are no longer inaccessible to these reagents. 相似文献
4.
1. In native egg albumin no SH groups are detectable, whereas in completely coagulated albumin as many groups are detectable as are found in the hydrolyzed protein. In egg albumin partially coagulated by heat the soluble fraction contains no detectable groups, and the insoluble fraction contains the number found after hydrolysis. 2. In the reversal of denaturation of serum albumin, when insoluble protein regains its solubility, S-S groups which have been detectable in the denatured protein, disappear. 3. When egg albumin coagulates at an air-water interface, all the SH groups in the molecule become detectable. 4. In egg albumin coagulated by irradiation with ultraviolet light, the same number of SH groups are detectable as in albumin coagulated by a typical denaturing agent. 5. When serum albumin is denatured by urea, there is no evidence that S-S groups appear before the protein loses its solubility. 6. Protein denaturation is a definite chemical reaction: different quantitative methods agree in estimates of the extent of denaturation, and the same changes are observed in the protein when it is denatured by different agents. A protein molecule is either native or denatured. The denaturation of some proteins can be reversed. 相似文献
5.
Alain F. Chaffotte Mario M. Zakin Michel E. Goldberg 《Biochemical and biophysical research communications》1980,92(2):381-388
In order to test if the α and β2 subunits of tryptophan synthetase and tryptophanase, three proteins involved in the metabolism of tryptophan in Escherichia coli K 12, have some common structural features reflecting an evolutionary filiation, an immunochemical comparison of these enzymes has been made using antibodies directed against either the native or the denatured β2 protein. The lack of cross-reactivity observed in the case of the three proteins studied, even when in their denatured state, suggests that, despite their functional relationships, they probably do not derive from a common ancestor. 相似文献
6.
When a denatured polypeptide is put into refolding conditions, it undergoes conformational changes on a variety of times scales. We set out here to distinguish the fast events that promote productive folding from other processes that may be generic to any non-folding polypeptide. We have apply an ab initio folding algorithm to model the folding of various proteins and their compositionally identical, random-sequence analogues. In the earliest stages, proteins and their scrambled-sequence counterparts undergo indistinguishable reductions in the extent to which they explore conformation space. For both polypeptides, an early contraction occurs but does not involve the formation of a distinct intermediate. Following this phase, however, the naturally-occurring sequences are distinguished by an increase in the formation of three-body correlations wherein a hydrophobic group desolvates and protects an intra-molecular hydrogen bond. These correlations are manifested in a mild but measurable reduction of the accessible configuration space beyond that of the random-sequence peptides, and portend the folding to the native structure. Hence, early events reflect a generic response of the denatured ensemble to a change in solvent condition, but the wild-type sequence develops additional correlations as its structure evolves that can reveal the protein's foldability. 相似文献
7.
A method for measuring the adsorption of dimethyl sulfoxide on native and denatured trypsin and albumin was developed. On native proteins, no positive adsorption was registered, and a slight negative adsorption within the limits of experimental error was observed. It was shown that the properties of denatured proteins depend on the mode and conditions of denaturation. On one of denatured trypsin specimens, positive adsorption of dimethyl sulfoxide was registered, on other specimens no adsorption was observed. The reason for this behavior lies in the hydrophobic nature of adsorption of dimethyl sulfoxide at the interface, while the surface of native protein globules and, probably, most denatured protein specimens is hydrophilic. 相似文献
8.
The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite. 相似文献
9.
The denaturation of hemoglobin by salicylate in neutral solution is completely reversible. There is a mobile equilibrium between native and denatured hemoglobin in neutral salicylate solution. The higher the salicylate concentration the greater is the percentage denaturation. When there is a mobile equilibrium between the native and denatured forms of a protein, denaturation is caused by the addition of any substance which has a greater affinity for the denatured than for the native form. Theoretically the heat of denaturation must vary with the denaturing agent and must depend on the heat of combination of the denaturing agent with the protein. 相似文献
10.
ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains. 相似文献
11.
A three-dimensional lattice model of protein designed to assimilate lysozyme is introduced. An attractive interaction is assumed to work between preassigned specific pairs of units, when they occupy the nearest-nighbor lattice points. The behavior of this lattice lysozyme is studied by a Monte Carlo simulation method. Because of the specific interunit interactions,“native state” of the lattice lysozyme is stable at low temperatures. Conformational fluctuations in the native state are observed to occur at both termini and loop regions of the main chain existing on the surface. The process of unfolding and denatured states of this model are discussed. Complete refolding from a denatured state was not observed. However, by starting from partially folded structures, the native conformation could be attained. From these observation it is concluded that, in the process of folding of proteins as simplified in a lattice model, nulceation is a rate-limiting factor. The artificial character of this model and possible improvement are discussed. 相似文献
12.
Basharov MA 《Indian journal of biochemistry & biophysics》2012,49(1):7-17
Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome. 相似文献
13.
Murata H Sakaguchi M Futami J Kitazoe M Maeda T Doura H Kosaka M Tada H Seno M Huh NH Yamada H 《Biochemistry》2006,45(19):6124-6132
Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available. 相似文献
14.
Protein structure by Fourier transform infrared spectroscopy: second derivative spectra 总被引:12,自引:0,他引:12
Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein. 相似文献
15.
Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA 总被引:14,自引:0,他引:14
We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites. 相似文献
16.
The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment. 相似文献
17.
Complexing and denaturation of DNA by methylmercuric hydroxide. II. Ultracentrifugation studies 总被引:2,自引:0,他引:2
When increasing concentrations of methylmercuric hydroxide are added to a Cs2SO4 solution of native DNA, the buoyant density of DNA is unaltered until a critical concentration is reached above which there is a cooperative transition to denatured DNA which now binds so much CH3HgOH that it becomes very dense and nonbuoyant. As increasing concentrations of methylmercuric hydroxide are added to a Cs2So4 solution of denatured DNA, the buoyant density gradually increases, indicating a gradual increase in the amount of methylmercury cation bound. The denatured DNA methylmercury complex becomes nonbuoyant at the same concentration of methylmercuric hydroxide as does the native DNA. These results support our previous interpretation that CH3HgOH reacts with the imino NH bonds of thymine and guanine in nucleic acids. The reaction occurs more or less independently at the different binding sites for denatured DNA, but it occurs cooperatively with simultaneous denaturation for native DNA. The nature of the transition of denatured DNA to the nonbuoyant state is not known, but it is probably due to an abrupt decrease in the degree of hydration of the DNA when its density and hydrophobic character are sufficiently increased by the binding of the methylmercury cation. Direct measurements of the amount of methylmercury bound by DNA, as observed by preparative ultracentrifugation, confirm approximately the buoyant density results as to the amount of methylmercury bound. The possibility of using methylmercuric hydroxide as a reagent for the separation of complementary strands, depending on then thymine of their thymine plus guanine content, is discussed. 相似文献
18.
Coarse-grained strategy for modeling protein stability in concentrated solutions. II: phase behavior
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We use highly efficient transition-matrix Monte Carlo simulations to determine equilibrium unfolding curves and fluid phase boundaries for solutions of coarse-grained globular proteins. The model we analyze derives the intrinsic stability of the native state and protein-protein interactions from basic information about protein sequence using heteropolymer collapse theory. It predicts that solutions of low hydrophobicity proteins generally exhibit a single liquid phase near their midpoint temperatures for unfolding, while solutions of proteins with high sequence hydrophobicity display the type of temperature-inverted, liquid-liquid transition associated with aggregation processes of proteins and other amphiphilic molecules. The phase transition occurring in solutions of the most hydrophobic protein we study extends below the unfolding curve, creating an immiscibility gap between a dilute, mostly native phase and a concentrated, mostly denatured phase. The results are qualitatively consistent with the solution behavior of hemoglobin (HbA) and its sickle variant (HbS), and they suggest that a liquid-liquid transition resulting in significant protein denaturation should generally be expected on the phase diagram of high-hydrophobicity protein solutions. The concentration fluctuations associated with this transition could be a driving force for the nonnative aggregation that can occur below the midpoint temperature. 相似文献
19.
Heike I. Rsner Martina Caldarini Gregory Potel Daniel Malmodin Maria A. Vanoni Alessandro Aliverti Ricardo A. Broglia Birthe B. Kragelund Guido Tiana 《Proteins》2022,90(1):96-109
The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process. 相似文献
20.
Giuseppe D’Alessio 《FEBS letters》2011,585(15):2403-2404
According to the structure-to-function paradigm proteins fold into a 3D structure for exerting their functions. Intrinsically destructured proteins with important biological functions have been identified and studied, but they assume a structure when interacting in the cell with their partners. There are instead bactericidal proteins, endowed also with other diverse activities (glycoside hydrolases, RNases, a defensin), which are lost when the proteins are denatured or inactivated, whereas the bactericidal activity is surprisingly conserved.The hypothesis is advanced that these proteins are not bactericidal per se, but because they store in their amino acid sequences peptide segments that display bactericidal activity when cut out as free peptides from the proteins. These bactericidal proteins would thus be merely containers of bactericidal peptides. 相似文献