Extent Of Conformational Changes Research Articles

Effect of surface hydrophobicity on the conformational changes of adsorbed fibrinogen

The extent of conformational changes of fibrinogen adsorbed on germanium, poly(hydroxyethyl methacrylate) (poly(HEMA), Biomer, and polystyrene surfaces was studied using Fourier transform infrared spectroscopy (FTIR) coupled with attenuated total reflectance (ATR) optics. The contact angles of water on the solid surfaces were 19.3°, 35.5°, 37.6°, and 90.5°, respectively. The adsorption kinetics of fibrinogen from 1 mg/ml solution showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. The spectra of fibrinogen in the bulk solution and of fibrinogen tightly adsorbed on germanium, poly(HEMA), Biomer, and polystyrene surfaces were treated using Fourier self-deconvolution and the synthetic single-peak fitting techniques to resolve the overlapped peaks in the amide I and II regions. It was found that some α-helical structures were changed into the unordered structures and the content of β-turns was increased upon the protein adsorption. A weighted-peak shift method was used to examine the extent of the protein conformational changes upon adsorption on hydrophobic and hydrophilic surfaces. The results indicated that the adsorbed fibrinogen underwent a larger degree of conformational changes as the surface hydrophobicity increased.

Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by α-chymotrypsin: A model system with preexistence of the isomerization equilibrium

The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine α-chymotrypsin have been investigated. In studies at pH 7 and 20 °C the presence of 0.5 m sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 °C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 m m) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.

Identification of a monoclonal antibody specific for a neoantigenic determinant on .alpha.2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational changes induced by hapten in murine monoclonal antibodies to dinitrophenyl groups - the analysis by temperature-perturbation spectroscopy

Two samples of murine monoclonal antibodies to dinitrophenyl groups were studied by difference thermal perturbation spectroscopy with particular attention to changes in the amount of perturbed chromophores induced in antibodies as a result of hapten binding (ε-2,4-dinitrophenyl- l-lysine). Despite the fact that both antibody samples belong to immunoglobulin Gl and have the same type of light chain, κ, they were found to differ significantly in the number of the chromophores perturbed by temperature. The binding of hapten decreases the perturbation of chromophores only in the sample with the less rigid structure, as regards thermal perturbation. These data provide evidence that differences in the rigidity of the structure of variable domains affect the extent of conformational changes induced in the antibodies due to the interaction with an antigen.

Mechanism of adenosinetriphosphatase activity of trinitrophenylated myosin subfragment 1.

The rate and extent of conformational changes induced by nucleotides in trinitrophenylated myosin subfragment 1 (S-1) were studied. The absorbance spectrum of the trinitrophenyl moiety attached to the reactive lysyl residue of S-1 changed considerably upon addition of ATP or its analogues. ATP or AMP-PNP induced a larger spectral change than that of ADP, and its value was independent of the presence of Mg2+. The trinitrophenylation of the reactive lysine considerably decreased the enhancement of the tryptophan fluorescence induced by MgATP, while it did not affect fluorescence enhancement induced by MgADP or MgAMP-PNP. The rate of formation of nucleotide-induced absorbance changes in trinitrophenylated (TNP) S-1 was followed in a stopped-flow spectrophotometer. The reaction could be described by a single exponential at every nucleotide concentration. The calculated apparent first-order rate constant, kobsd, increased with rising concentrations of MgATP, ATP, MgADP, and MgAMP-PNP and finally reached a plateau. The absorbance change that appeared upon addition of MgATP to TNP S-1 eventually decayed in two phases until it reached the level of that induced by MgADP. The nucleotide concentration dependence of kobsd deviated from a hyperbolic function in all cases studied. The nucleotide concentration dependence of the signal amplitude was also measured, and it was found to be independent of the presence of Mg2+. The obtained results deviated from a computer-simulated binding curve, which was computed by assuming one identical binding site for each TNP S-1 molecule. The results indicate that an enzyme-substrate complex is the predominant intermediate in the TNP S-1 catalyzed MgATP hydrolysis, instead of an enzyme-product complex, which is the case with normal, unmodified S-1.

The structure and function of the third component of human complement—I. The nature and extent of conformational changes accompanying C3 activation

The nature and extent of conformational changes accompanying the enzymatic activation of C3 were investigated using spectroscopic methodology. Techniques sensitive to changes in backbone conformation (e.g. far UV circular dichroism (CD)) or to the perturbation of a considerable proportion of the aromatic residues in the protein (e.g., intrinsic tryptophan fluorescence or solvent perturbation difference spectroscopy) failed to show substantial spectral differences following the activation process. In contrast, methods capable of detecting changes in the local environment of a small number ofchromophores show significant spectral alterations following C3 activation. For example, the ultraviolet difference spectrum of a C3a + C3b digestion mixture versus native C3 provides evidence for the net burial of approximately one tryptophan residue upon activation ( Δε 294 = + 1400) while the negative direction of the difference spectrum below 288 nm is suggestive of a net exposure of a small number of tyrosine residues. Peptide cleavage induced changes in the local environment of aromatic residues are also evident in comparing the near UV (330-250 nm) CD spectra of pre- and post-activation C3. Part of the difference observed in the 270-250 nm region may result from an altered environment of disulfide bonds. This is further suggested by our findings that disulfides are more resistant to reduction in C3b than in the native molecule. Finally, we report that the emission spectrum due to the binding of the fluorescent compound 1-anilino-8-naphthalene sulfonate (ANS) to C3 displays a very pronounced enhancement following C3 conversion. This finding suggests that there are substantial changes in the surface hydrophobicity of at least a portion of the C3 molecule following its activation. Taken together, the conformational probes described in this study provide a foundation for investigating the dynamic processes occurring in C3 following its activation.

Structure of functional a salina - E Coli hybrid ribosome by electron microscopy

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination -- small 30S E coli and large 60S A salina -- fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A slina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.

Hydrogen exchange of dark-adapted and illuminated bacteriorhodopsin

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Electron spin exchange in rigid biradicals

Preparation is reported for six nitroxyl biradicals in which the extent of conformational change is strongly limited by the rigidity of the structure connecting the radical groups. S resonances were observed for all reported biradicals and were used to calculate the exchange energy (J) from the separation ..delta..H between the strongest S resonance and the center of the spectrum: J = ..delta..H-a/sup 2//..delta..H where a is the hyperfine splitting due to nitroxyl nitrogen. Values of J ranging from 6 to 172 G were determined. The solvent effects on the exchange energy was reported for hexane, xylene, acetone, DMSO, chloroform, and methanol. (DDA)

Analysis of conformationally-linked dissociation of proteins.

Conformationally-linked dissociation equilibria of dimeric proteins have been examined to determine how experimentally obtainable parameters, such as the apparent dissociation constant, k D , and the apparent conformational transition constant, K conf , are related to intrinsic subunit interaction constants, K A or K B , and intrinsic isomerization constants, K 1 or k 2 . Limiting models are considered in which either the conformational change occurs before dissociation or in which the dissociation occurs before the conformational change, as well as a general model including both possibilities. Models are also considered in which three conformations are allowed or in which four subunits (tetramers) are involved. Simulated data for the dimer equilibria are presented to show how variation of protein concentration and variation of certain constants affect the proportion of various molecular species, the weight-average molecular weight, and the overall extent of conformational change. Methods are suggested to distinguish between the different limiting cases based upon the dependence of K D and/or K conf on protein concentration, perturbant concentration, and temperature. It is concluded that methods used to calculate self-dissociation constants oligomeric proteins include linked isomerization reactions such that the equilibrium constant obtained should not be considered as a true subunit interaction term. Indeed, dissociation can occur under the influence of a perturbant with no change in the intrinsic affinity of the subunits but with the sole effect of the perturbant being on a linked conformational change. Additional experiments on the thermodynamics of the conformational change are required to determine the actual relationship. Depending on the complexity of the equilibria involved and the relative value of the equilibrium constants, the extent of the conformational change can vary directly with, vary inversely with, or he independent of the total protein concentration. Even when intrinsic subunit affinities are not affected by the perturbant, the extent of conformational change can vary with protein concentration. Interpretation of data from proteins which may be involved in conformationally-linked dissociation reactions, therefore, must be made with caution.

The effect of tolerance on the specificity of the antibody response and on immunogenicity. Antibody response to conformationally and chemically altered antigens.

Animals were rendered tolerant to human albumin and were then immunized with azo derivatives of human albumin which differed in the number of hapten groups per molecule and in the extent of conformational change. The incidence and specificity of the resulting antibody response was studied and the presence of antibody to azo groups and to conformationally altered protein determinants was demonstrated. Reactivity with the tolerance-inducing antigen was shown to be due to antibodies directed against conformationally altered protein determinants. The difference in the response of tolerant animals to hapten-poor and hapten-rich derivatives was attributed to the extent of conformational alteration. A genetic factor appeared to be implicated in the capacity of tolerant animals to respond to an antigen which cross-reacts with tolerance-inducing macromolecules.

Stability of structures of human γ-globulin

A homogeneous fraction with an s 20, w value of 6.6 obtained from human γ-globulin by separation on a Chromatographic column was studied. After reduction with 0.1 m mercaptoethanol, 6–8 sulfhydryl groups were found on titration. On acidification of solutions of the native protein, irreversible changes in the difference spectra appeared at pH 3 with maximum differences in extinction at 250 and 280 nm. On changing the dielectric constant of the solvent (ethylene glycol-water solutions) no change in structure was observed. However, thermal denaturation was accompanied by an increase in the stability of the structure. On the contrary, solutions of the reduced human γG-globulin were found to undergo thermally induced variation of conformation. The extent of conformational change was found to depend on the extent of reduction of the disulfide bonds and to be suppressed in high ionic strength solutions. A viscosity increase which appeared before the denaturation itself was also observed on heating. It was not influenced by changes in pH or by reduction of the disulfide bonds with mercaptoethanol.