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1.
The influence of proline cis-trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two-state transition model well. The temperature dependencies of kuf and kf over a wide temperature range showed that ΔC = 0 and ΔC = ?6.7 kJ K?1 mol?1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol?1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol?1 is primarily required for the disruption of long-range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.  相似文献   

2.
Wei Liu  Takashi Norisuye 《Biopolymers》1988,27(10):1641-1654
Weight-average molecular weights Mw, second virial coefficients, and z-average radii of gyration 〈S2〉 were determined by light scattering as a function of temperature T for four sodium salt samples of xanthan in 0.01M aqueous NaCl, in which the polysaccharide undergoes an order–disorder conformation change with increasing T. The data for 〈S2〉 and Mw at 25 and 80°C, the lowest and highest temperatures studied, confirmed the previous conclusion that the predominant conformation at the former T, i.e., in the ordered state, is a double helix, while that at the latter T, i.e., in the disordered state, is a dimerized coil expanded by electrostatic repulsions between charged groups of the polymer. As T was increased from 25 to 80°C, 〈S2〉 sigmoidally decreased or increased depending on the dimer's molecular weight. This temperature dependence of 〈S2〉 and that determined elsewhere for a high molecular weight sample were found to be described almost quantitatively by a simple dimer model in which the double helix melts from both ends, when the double-helical fraction in the dimer at a given T estimated previously from optical rotation data was used.  相似文献   

3.
The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P2O, P3O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH?, Cl?, F?, H2PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).  相似文献   

4.
5.
We describe conditions which lead to complete helix formation of poly(I) in the presence of NH. Binding of NH is shown to be specific in the presence of Li+, which does not by itself support helix formation under these conditions. The NH–poly(I) complex is characterized by uv, CD, and ir spectroscopy. The CD spectrum is strikingly different from those of the Na+ or K+ complexes, the first extremum being changed from negative for the metal ions to positive for NH. A stereospecific model is proposed for the NH–poly(I) helix in which the N of NH is located on the axis of the four-stranded helix, midway between planar tetramers formed by the bases. The model is consistent with the tetrahedral symmetry of NH, the requirement for four acceptable hydrogen bonds, the observed stability of the helix, and the accepted geometry of the backbone.  相似文献   

6.
A Cabani  A Paci  V Rizzo 《Biopolymers》1976,15(1):113-129
Using the formalism of nearest-neighbor Ising model and assuming that the allowed states for a monomeric unity of a polypeptide chain in solutions containing strong acids are E (helix), C (coil), and CS (solvent-bonded coil), the partition function of the system was deduced analytically. Equations were obtained which permitted the prediction of the characteristic thermodynamic behavior of the helix–coil transition under these conditions. These equations were used to examine critically the possible correlations between experimental data obtained using different techniques. Particular attention was devoted to quantities called “transition enthalpies,” obtained from the slope of the transition curves at the point where the helix fraction is one-half (ΔH), or for measurements of the heat of solution of the polymer over the total range of solvent composition (ΔH), or from heat capacity measurements taken at various temperatures (ΔH). Literature data of ΔH(j = opt, sol, cal) for the system poly-γ-benzyl-L -glutamate in mixtures of dichloroacetic acid and 1,2-dichloroethane were carefully analyzed.  相似文献   

7.
Conformation and folding in histones H1 and H5   总被引:1,自引:0,他引:1  
Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a Tm and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.  相似文献   

8.
Empirical force-field calculations and ir and 1H-nmr spectra indicate that five-membered (C5) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C5/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.  相似文献   

9.
The kinetics of binding of the cationic surfactant cetyltrimethyl ammonium bromide with the Na salt of carboxymethyl cellulose was studied by the electrometric method using cetyltrimetlyl ammonium+ (CTA+) ion-selective polyvinyl chloride membrane electrode. The binding process followed the first-order kinetics and occurred in three stages. Its affinity increased with increasing CTA bromide concentration and decreased with ionic strength. The activation process comprised moderate E and ΔH and negative ΔS for all three stages with a ΔH < TδS trend proving it to be entropy controlled. The ΔG values followed the trend ΔG < ΔG < ΔG (in accordance with k1 > k2 > k3). The enthalpies (ΔH) and entropies (ΔS) of activation followed a systematic and interdependent trend. The multiple-stage binding kinetics is grossly comparable with the kinetics of binding of proteins to solid surfaces. © 1995 John Wiley & Sons, Inc.  相似文献   

10.
Small-angle x-ray scattering of poly(γ-methyl-L -glutamate), [Glu(OMe)]n, in m-cresol and in pyridine was measured to determine the mass per unit length, Mq, and the radius of gyration of the cross section, 〈S1/2. It was confirmed from the values of Mq that [Glu(OMe)]n exists in an α-helical conformation in these solvents. It was elucidated from the calculations on 〈S1/2 that the side chains come in moderately close contact with the main chain in these solvents. It was indicated from the analysis of the outer portion of the scattering curves that the side-chain conformation varied depending on the solvent.  相似文献   

11.
The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k1 + k?1) and k (= k2 + k?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.  相似文献   

12.
Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with Cp2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.  相似文献   

13.
We studied the interactions of the substitution-inert inversion-labile complexes Fe(bipy) and Fe(phen) [and the inversion-stable complex Ru(bipy)] with DNA. The association of these complexes to DNA is mainly electrostatic, and Fe(phen) shows a more effective binding to DNA than the two bipyridyl complexes, possibly owing to a different binding mode. The interactions are enantioselective, leading to a Pfeiffer shift in the diastereomeric inversion equilibria and an excess of the Δ-enantiomer of Fe(phen) and Fe(bipy), which is directly monitorable through CD. The partition constants for the inversion equilibrium range from 1.3 to 2.0 for Fe(bipy) and Fe(phen), depending on ionic conditions. From flow LD information about the orientation of the complexes on DNA was obtained: it is consistent with a fit of the Δ-enantiomer in the major groove of the right-handed DNA helix. The mechanisms of interaction are discussed against equilibrium, spectroscopic, and kinetic data.  相似文献   

14.
Four fundamental Raman lines were observed at 159, 111, 55 and 27 cm-1 corresponding to the I bound (I) in amyloses with DP from 20 to 100, regardless of the degree of polymerization of I and the excitation wavelength. The spectral resolution was based on the molar extinction coefficient and molar ellipticity spectra of I. Eight bands, named, S1, S2, ?, S8 from long to short wavelength, were isolated. These were found regardless of the DP. By a resonance excitation Raman study, the characteristics of S3 and S4, comprising the shoulder around 480 nm, were found to be different from those of S1 and S2, comprising the blue band. The assignment of the spectra was based on the electronic states of the monomeric I in the exciton-coupled dimeric unit. It was concluded that the blue band (S1,S2) belonged to the long-axis transitions and the shoulder band (S3,S4) to the short-axis ones on the monmeric coordinate system.  相似文献   

15.
Densities of solutions of several α-amino acids and peptides in 3 and 6m aqueous urea solvents have been determined at 298.15 K. These data have been used to evaluate the infinite-dilution apparent molar volumes of the solutes and the volume changes due to transfer (V ) of the α-amino acids and peptides at infinite dilution from water to aqueous urea solutions. The sign and magnitude of the V values have been rationalized in the framework of Friedman's cosphere-overlap model. The V values for the glycyl group (? CH2CONH? ) and alkyl side chains have been estimated.  相似文献   

16.
The compositional buoyant densities, ρ;, of human γ-immunoglobulin, bovine serum mercaptalbumin, and egg albumin have been measured in CsCl solutions in the analytical ultracentrifuge as a function or pressure. Standard pressure coefficients, ψ0, and standard partial specific volumes of the solvated proteins, υ ,0, have been computed from these data. The ψ0 values obtained are strikingly different from each other and from the only other pressure coefficients which have been measured, those values obtained for nucleic acids and nucleoproteins. The ψ value for γ-immunoglobulin is negative, the first nonpositive value obtained, and suggests an unusual internal structure for this protein. The pressure coefficient of mercaptalbumin is not constant. A second-order relation is derived and utilized to interpret these data. The slope of the ρ(P) plot for egg albumin was constant and negative and yielded values of ψ0 which are about 20% as large as those reported for DNA. Evaluation of published isopiestic data for egg albumin in CsCl solutions provided the dependence of preferential hydration on water activity. This quantity, (dΓ′/da) as well as α, were found to be negative. The values of ψ0 and α were used to compute the effective density gradient from which the correct molecular weight of egg albumin was obtained. The apparent specific volume of egg albumin in a buoyant CsCl solution was measured using the Mettler-Paar densimeter.  相似文献   

17.
Molecular mechanics calculations have been used to determine the preferred physical association sites of the known alkylating agent dimethyl aziridinium ion (Az+) and a CH prototype test probe with B-form, tetrameric DNA sequences. Electrostatic interactions are most important in determining these preferential physical association sites. In turn, the intermolecular energy minima depend on the charge distribution assigned to the DNA sequence. However, for three reported DNA charge distributions, only two distinct sets of energy minima were obtained for the CH-like ion interacting with (G-C)4, (A-T)4, and [(G-C)·(A-T)]2 deoxyribonucleic acids. These minima correspond to physical association geometries in which the CH-like ion is near known alkylation sites. The results of the Az+ … [(G-C)·(A-T)]2 interaction are virtually identical to those found for the CH-like ion. Aqueous solvation energetics have little effect on the physical association of Az+ with [(G-C)·(A-T)]2.  相似文献   

18.
If the collagen triple helix is so built as to have one set of NH ? O hydrogen bonds of the type N3H3(A) ? O2(B), then it is possible to have a linkage between N1H1(B) and O1(A) through the intermediary of a water molecule with an oxygen O leading to the formation of the hydrogen bonds N1(B) ? O and O (A). In the same configuration, another water molecule with an oxygen O can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O O1(A) and O O0 (B). The two water oxygens also become receptors at the same time for CH ? O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ? O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.  相似文献   

19.
Spectrophotometric techniques have been employed to study the binding of bromophenol red (BPR) to hen egg white lysozyme and the consequent inhibition of enzyme activity. Experimental evidence is given from the dye binding studies in the presence of hexasaccharide and from the studies on activity that BPR binds at a site outside the proposed cleft region (A–F) in such a way that it inhibits the lytic activity towards cell walls but does not inhibit the activity towards hexasaccharide. These observations are consistent with the kinetics of binding [studied using temperature-jump (T-jump)] in the presence of Co++ or chitotriose in large concentrations and the experiments with acetylated lysozyme which suggest that the binding site of BPR is closer to a lysine residue near the cleft. It is suggested that the binding site of BPR could be important in positioning the peptide segment of the cell walls, which are cleaved in the cleft. Evidence for the statement that this binding takes place at least by a two-step process, in which the bimolecular step is followed by a slower monomolecular step, is given from the observations of two types of 1:1 complexes at 24°C in equilibrium studies and from the concentration dependence of the relaxation observed at 605 nm in the T-jump experiments. The binding process is examined by analyzing the T-jump data obtained between 18 and 33°C in the pH range 5.2–9.2 and ionic strength 0.01–01. The ionic strength and pH dependences of the equilibrium constant associated with the bimolecular step k2/k1 and the forward rate constant associated with monomolecular step k3 have been given as evidence for the suggestion that a Coulombic interaction is involved in the first step of binding. However, the final state of binding is hydrophobic in nature. The enthalpy of activation ΔH and the entropy of activation ΔS associated with kf[= k3(k1/k2)] showed compensation behavior with pH variation, with maxima around pH ~ 7.5 in H2O. This has been interpreted as a maximal disordering of water structure in a region of the enzyme at this pH during the monomolecular step. However, the binding of chitotriose or Co++ in the cleft reduces the ΔH and ΔS associated with the monomolecular step of BPR binding, probably by disordering the structured water during their binding in the cleft. The differences in the kinetic parameters obtained in H2O and in D2O probably arise due to subtle differences in the conformation of the enzyme in the two solvents and apart from isotope effects. The correlation between the pH (or pD) dependence of the “intrinsic activity” towards cell walls and ΔH or ΔS indicates that ordered water structure could be playing a role in controlling the catalytic activity. It is also suggested that this factor is associated with the rate constant k3s of the monomolecular step leading to the formation of the final bound state of the substrate in cell lysis, which is also a factor controlling kcat.  相似文献   

20.
Poly(L -arginine) assumes the α-helix in the presence of the tetrahedral-type anions or some polyanions by forming the “ringed-structure bridge” between guanidinium groups and anions which is stabilized by a pair of hydrogen bonds and electrostatic interaction [Ichimura, S., Mita, K. & Zama, M. (1978) Biopolymers 17 , 2769–2782; Mita, K., Ichimura, S. & Zama, M. (1978) Biopolymers 17 , 2783–2798]. This paper describes the parallel CD studies on the conformational effects on poly (L -homoarginine) of various mono-, di-, polyvalent anions and some polyanions, as well as alcohol and sodium dodecylsulfate. The random coil to α-helix transition of poly(L -homoarginine) occurred only in NaClO4 solution or in the presence of high content of ethanol or methanol. The divalent and polyvalent anions of the tetrahedral type (SO, HPO, and P2O), which are strong α-helix-forming agents for poly(L -arginine), failed to induce the α-helical conformation of poly(L -homoarginine). By complexing with poly(L -glutamic acid) or with polyacrylate, which is also a strong α-helix-forming agent for poly(L -arginine), poly(L -homoarginine) only partially formed the α-helical conformation. Monovalent anions (OH?, Cl?, F?, and H2PO) did not change poly(L -homoarginine) to the α-helix, and in the range of pH 2–11, the polypeptide remained in an unordered conformation. In sodium dodecylsulfate, poly(L -homoarginine) exhibited the remarkably enlarged CD spectrum of an extended conformation, while poly(L -arginine) forms the α-helix by interacting with the agent. Thus poly(L -homoarginine), compared with poly(L -arginine), has a much lower ability to form the α-helical conformation by interacting with anions. The stronger hydrophobicity of homoarginine residue in comparison with the arginine residue would provide unfavorable conditions to maintain the α-helical conformation.  相似文献   

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