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.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang XD
PROVIDER: S-EPMC1218615 | biostudies-other | 1997 Aug
REPOSITORIES: biostudies-other
Similar Datasets
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.
| S-EPMC3780615 | biostudies-literature
Project description:The Ca2+-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb3+ is commonly used as a luminescent Ca2+ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca2+ and the lanthanides in CaM. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca2+-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca2+-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.
| S-EPMC5889669 | biostudies-literature
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.
| S-EPMC6034165 | biostudies-literature
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.
| S-EPMC2790481 | biostudies-literature
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.
| S-EPMC3770747 | biostudies-literature
Project description:Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance (13)C and high-resolution (of 0.01 Hz order) (1)H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal NH(3)(+) group in achatin-I; the de-ionization of NH(3)(+) decreases the bound fraction of the peptide from approximately 15% to nearly none. The electrostatic attraction between the N-terminal positive NH(3)(+) group and the negative PO(4)(-) group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the (13)C chemical shifts (delta) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Delta delta of the (13)C resonance for almost all the carbon sites in achatin-I. The changes Delta delta for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic CO(2)(-) groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic d-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.
| S-EPMC1304359 | biostudies-literature
Project description:Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general "systems level" mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step.
| S-EPMC4169960 | biostudies-literature
Project description:The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys-66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys-66 is located. Above 2 kbar pressure at these pH values the protein with Lys-66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys-66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys-66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure.
| S-EPMC3427779 | biostudies-literature
Project description:The actin crosslinking protein filamin A (FLNa) mediates mechanotransduction, a conversion of mechanical forces into cellular biochemical signals to regulate cell growth and survival. To provide more quantitative insight into this process, we report results using magnetic tweezers that relate mechanical force to conformational changes of FLNa immunoglobulin-like repeats (IgFLNa) 20-21, previously identified as a mechanosensing domain. We determined the force magnitudes required to unfold previously identified structural organizations of the ?-strands in the two domains: IgFLNa 20 unfolds at ~15?pN and IgFLNa 21 unfolding requires significantly larger forces. Unfolded domain IgFLNa 20 can exist in two different conformational states, which lead to different refolding kinetics of the IgFLNa 20 and imply a significant impact on the reformation of the domain pair at reduced force values. We discuss the relevance of the findings to force bearing and mechanosensing functions of FLNa.
| S-EPMC3622079 | biostudies-other
Project description:Major depression is nearly twice as prevalent in women compared to men. In bipolar disorder, depressive episodes have been reported to be more common amongst female patients. Furthermore, periods of depression often correlate with periods of hormonal fluctuations. A link between hormone signaling and these mood disorders has, therefore, been suggested to exist in many studies. Estrogen, one of the primary female sex hormones, mediates its effect mostly by binding to estrogen receptors (ERs). Nuclear ERs function as transcription factors and regulate gene transcription by binding to specific DNA sequences. A nucleotide change in the binding sequence might alter the binding efficiency, which could affect transcription levels of nearby genes. In order to investigate if variation in ER DNA-binding sequences may be involved in mood disorders, we conducted a genome-wide study of ER DNA-binding in patients diagnosed with major depression or bipolar disorder. Association studies were performed within each gender separately and the results were corrected for multiple testing by the Bonferroni method. In the female bipolar disorder material a significant association result was found for rs6023059 (corrected p-value = 0.023; odds ratio (OR) 0.681, 95% confidence interval (CI) 0.570-0.814), a single nucleotide polymorphism (SNP) placed downstream of the gene coding for transglutaminase 2 (TGM2). Thus, females with a specific genotype at this SNP may be more vulnerable to fluctuating estrogen levels, which may then act as a triggering factor for bipolar disorder.
| S-EPMC3289647 | biostudies-literature