Perturbation of the antigen-binding site and staphylococcal protein A-binding site of IgG before significant changes in global conformation during denaturation: an equilibrium study.
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ABSTRACT:
SUBMITTER: Wang XD
PROVIDER: S-EPMC1218615 | biostudies-other | 1997 Aug
REPOSITORIES: biostudies-other
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Project description:A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.
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Project description:To investigate the cyanylated cysteine vibrational probe group's ability to report on binding-induced changes along a protein-protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.
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Project description:PurposeTransforming growth factor beta-induced protein (TGFBIp) aggregates into the phenotypic amyloid fibrils and/or non-amyloid deposits in corneal dystrophies and other disorders. While significant progress has been made in molecular genetics to successfully establish the link between the missense mutations of TGFBI and TGFBIp-related corneal dystrophies, the underlying mechanism for the abnormal aggregation remains elusive due to the lack of insights into the conformational perturbations induced by mutations. In the present study, we examined the effects of denaturants and a co-solvent on recombinant TGFBIp, with a focus on protein conformational changes and amyloid fibril formation.MethodsRecombinant TGFBIp was subjected to various spectroscopic studies, such as far-ultraviolet circular dichroism (far-UV CD), intrinsic tryptophan fluorescence and quenching, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, under various denaturing conditions (urea and guanidine hydrochloride [GndHCl], acidic pH, and trifluoroethanol [TFE, co-solvent]). A thioflavin T (ThT) fluorescence assay was used to determine the fibril formation of TGFBIp. In addition, a rabbit polyclonal antibody against the oligomer precursors that initiate the formation of amyloid fibrils was also used in dot blot experiments to detect the formation of prefibrillar precursors.ResultsThe purified recombinant TGFBIp is in the folded state according to its intrinsic tryptophan fluorescence analyses. A single-step unfolding process was observed in the GndHCl denaturation experiment. Results from far-UV CD, intrinsic tryptophan fluorescence, and ANS fluorescence experiments showed that TFE exerted its solvent effects by initially unfolding and transforming TGFBIp to a beta-sheet-enriched conformer at 20%. When increased to 40%, TFE changed TGFBIp into a non-native alpha-helix conformer. Although GndHCl and TFE led to protein unfolding, enhanced fibril formation could only be observed in the presence of TFE and at acidic pH, according to the ThT fluorescence assays. The paradigmatic protofibrillar TGFBIp oligomers were also detected during the fibril formation by the dot blot experiment.ConclusionsOur results suggest that protein unfolding may serve as the prerequisite but is not sufficient for the fibrillogenesis. Other factors, such as the solvent used, fragmentation, or pH, may also be crucial for the formation of TGFBIp fibrils.
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Project description:Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.2. Within experimental error the values of [Formula: see text] and mGuHCl for the various proteins measured over this wide range of pH maintain a constant offset from one another, tracing a series of approximately parallel curves. This data offers an independent means of determining the error of stabilities and slopes determined by guanidine hydrochloride denaturations and shows that previous error estimates are accurate. More importantly, this behavior cannot be reconciled with a three-state denaturation model for staphylococcal nuclease. The large variations in mGuHCl observed in these mutants must therefore arise from other causes.
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