Abani K. Bhuyan

On the mechanism of SDS-induced protein denaturation.

To understand the mechanism of ionic detergent-induced protein denaturation, this study examines the action of sodium dodecyl sulfate on ferrocytochrome c conformation under neutral and strongly alkaline conditions. Equilibrium and stopped-flow kinetic results consistently suggest that tertiary structure unfolding in the submicellar and chain expansion in the micellar range of SDS concentrations are the two major and discrete events in the perturbation of protein structure. The nature of interaction between the detergent and the protein is predominantly hydrophobic in the submicellar and exclusively hydrophobic at micellar levels of SDS concentration. The observation that SDS also interacts with a highly denatured and negatively charged form of ferrocytochrome c suggests that the interaction is independent of structure, conformation, and ionization state of the protein. The expansion of the protein chain at micellar concentration of SDS is driven by coulombic repulsion between the protein-bound micelles, and the micelles and anionic amino acid side chains.

Backbone dynamics of barstar: A 15N NMR relaxation study

Backbone dynamics of uniformly 15N‐labeled barstar have been studied at 32°C, pH 6.7, by using 15N relaxation data obtained from proton‐detected 2D {1H}‐15N NMR spectroscopy. 15N spin‐lattice relaxation rate constants (R1), spin‐spin relaxation rate constants (R2), and steady‐state heteronuclear {1H}‐15N NOEs have been determined for 69 of the 86 (excluding two prolines and the N‐terminal residue) backbone amide 15N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model‐free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (τm), the generalized order parameter (S2), the effective correlation time for internal motions (τe), and NH exchange broadening contributions (Rex) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D‖/D⟂) is 0.82 ± 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, ωH, and ωN, where ωH,N are proton and nitrogen Larmor frequencies. The value of τm obtained from model‐free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The τm determined here is different from that calculated previously from time‐resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 ± 0.02. A comparison of the order parameters with the X‐ray B‐factors for the backbone nitrogens of wild‐type barstar does not show any considerable correlation. Model‐free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s−1, indicating the presence of conformational averaging motions only for a small subset of residues. Proteins 2000;41:460–474.

Unfolding action of alcohols on a highly negatively charged state of cytochrome C.

It is well-known that hydrophobic effect play a major role in alcohol-protein interactions leading to structure unfolding. Studies with extremely alkaline cytochrome c (U(B) state, pH 13) in the presence of the first four alkyl alcohols suggests that the hydrophobic effect persistently overrides even though the protein carries a net charge of -17 under these conditions. Equilibrium unfolding of the U(B) state is accompanied by an unusual expansion of the chain involving an intermediate, I(alc), from which water is preferentially excluded, the extent of water exclusion being greater with the hydrocarbon content of the alcohol. The mobility and environmental averaging of side chains in the I(alc) state are generally constrained relative to those in the U(B) state. A few nuclear magnetic resonance-detected tertiary interactions are also found in the I(alc) state. The fact that the I(alc) state populates at low concentrations of methanol and ethanol and the fact that the extent of chain expansion in this state approaches that of the U(B) state indicate a definite influence of electrostatic repulsion severed by the low dielectric of the water/alcohol mixture. Interestingly, the U(B) ⇌ I(alc) segment of the U(B) ⇌ I(alc) ⇌ U equilibrium, where U is the unfolded state, accounts for roughly 85% of the total number of water molecules preferentially excluded in unfolding. Stopped-flow refolding results report on a submillisecond hydrophobic collapse during which almost the entire buried surface area associated with the U(B) state is recovered, suggesting the overwhelming influence of hydrophobic interaction over electrostatic repulsions.

Expansion and Internal Friction in Unfolded Protein Chain

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome c (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction.

Hydrophobic collapse overrides Coulombic repulsion in ferricytochrome c fibrillation under extremely alkaline condition.

Tuning of both hydrophobic and electrostatic interactions is thought to be important for the initial nucleation and stability of protein aggregates that self-assemble to produce amyloid fibrils. Importance of a critical balance of these two interactions has indeed been determined under various solution conditions of fibrillation, the acidic pH, in particular. To find out if fibrillar protein structures could be obtained under extreme alkaline conditions, cytochrome c was allowed to fibrillate in 0.1 N NaOH at 50 or 60 °C. Fibers do grow in alkali, but the fibrillation process depends little on the ionic strength of the solution. Illustrative fibril morphology readily obtained even in the absence of solvent cations poses the question as to how the severity of electrostatic repulsions is overcome to initiate aggregation. It appears that intermolecular hydrophobic collapse is so overwhelming that electrostatic repulsions are subdued, and the negative charges on protein molecules are relocated in a way conducive to fiber growth. This proposal seems consistent with computer simulation studies indicating central role of hydrophobic interactions. Morphologically, branched fibrils characterized by a wide distribution of diameter are assembled by winding two or more protofibrils. The results should guide selection of model parameters in theoretical studies of fibrillation.

Viscosity Dependence of Some Protein and Enzyme Reaction Rates: Seventy-Five Years after Kramers.

Kramers rate theory is a milestone in chemical reaction research, but concerns regarding the basic understanding of condensed phase reaction rates of large molecules in viscous milieu persist. Experimental studies of Kramers theory rely on scaling reaction rates with inverse solvent viscosity, which is often equated with the bulk friction coefficient based on simple hydrodynamic relations. Apart from the difficulty of abstraction of the prefactor details from experimental data, it is not clear why the linearity of rate versus inverse viscosity, k ∝ η(-1), deviates widely for many reactions studied. In most cases, the deviation simulates a power law k ∝ η(-n), where the exponent n assumes fractional values. In rate-viscosity studies presented here, results for two reactions, unfolding of cytochrome c and cysteine protease activity of human ribosomal protein S4, show an exceedingly overdamped rate over a wide viscosity range, registering n values up to 2.4. Although the origin of this extraordinary reaction friction is not known at present, the results indicate that the viscosity exponent need not be bound by the 0-1 limit as generally suggested. For the third reaction studied here, thermal dissociation of CO from nativelike cytochrome c, the rate-viscosity behavior can be explained using Grote-Hynes theory of time-dependent friction in conjunction with correlated motions intrinsic to the protein. Analysis of the glycerol viscosity-dependent rate for the CO dissociation reaction in the presence of urea as the second variable shows that the protein stabilizing effect of subdenaturing amounts of urea is not affected by the bulk viscosity. It appears that a myriad of factors as diverse as parameter uncertainty due to the difficulty of knowing the exact reaction friction and both mode and consequences of protein-solvent interaction work in a complex manner to convey as though Kramers rate equation is not absolute.

Cloning, Escherichia coli expression, purification, characterization, and enzyme assay of the ribosomal protein S4 from wheat seedlings (Triticum vulgare).

S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin).

The Off-Pathway Status of the Alkali Molten Globule Is Unrelated to Heme Misligation and Trans-pH Effects: Experiments with Ferrocytochrome c

The relevance of the alkali molten globule (B-state) to the folding pathway of cytochrome c has been studied further with the reduced state of the protein for which the B-state is prepared by ligating the ferrous heme iron with extrinsic CO under 1 mM gas concentration in the presence of NaCl. The CO derivative of ferrocytochrome c is desirable not only because it abrogates the interference of non-native heme ligands but the GdnHCl-unfolded protein refolds fast without deviating from the intrinsic folding pathway of the protein. Interstate folding-unfolding kinetics at alkaline and neutral pH conditions reveal that the B-state chain initially expands in the submillisecond regime in order to fold correctly to the native state. In this sense, it is an off-pathway nonproductive species which must dissipate some non-native elements before proceeding to fold. It is concluded that the alkali molten globule does not correspond to any possible transient structure in the folding pathway of cytochrome c.

Bacterially expressed recombinant WD40 domain of human Apaf-1

The apoptotic protease activating factor (Apaf-1) is a protein that binds to cytochrome c, and in the presence of dATP/ATP oligomerizes to assume the role of an adaptor platform for activating the caspase-9 zymogen. In order to study the biochemical and structural details of Apaf-1 function, we have generated an expression construct from pcDNA 3-Apaf-1XL for production of the WD40 domain ((WD40)Apaf-1) in Escherichia coli. The WD40 domain expressed contains 825 amino acids in addition to an N-terminal His(6) tag derived from the cloning vector. The expressed protein is invariably localized in the inclusion body fraction of E. coli. A simple protocol involving Sephadex G100 chromatography developed for purifying the protein starting from inclusion bodies has allowed protein recovery in highly pure form. Basic fluorescence and CD spectra indicate that the refolded protein has extensive secondary and tertiary structures. Immunoprecipitation studies have provided qualitative information about the binding interaction of (WD40)Apaf-1 and cytochrome c. The binding interaction has been quantified by spectrophotometric titration of cytochrome c with recombinant (WD40)Apaf-1. The results demonstrate a weak binding for cytochrome c and (WD40)Apaf-1 interaction, the binding affinity being 390 nM. The analysis indicates a 2:1 or possibly even 3:1 stoichiometry for cytochrome c and (WD40)Apaf-1 binding interaction.

Nonlinear effect of GdnHCl on hydration dynamics of proteins: a 1H magnetic relaxation dispersion study.

Proton magnetic relaxation dispersion investigations with aqueous solutions of lysozyme and bovine serum albumin (BSA) in the 0-5 M range of guanidine hydrochloride (GdnHCl), pH 4.4, 27 degrees C, were taken up with the objective of examining the hydration dynamics of internal cavity waters as the protein is held under increasingly destabilizing conditions. Field cycling NMR and conventional pulsed NMR techniques were employed to cover a frequency range of 100 kHz to 50 MHz. Analyses of dispersion profiles at different concentrations of GdnHCl were carried out considering the contributions from internal and surface waters. The denaturant-dependent variation of internal water contribution indicates that the reorientational disorder of internal waters decreases with increments of the denaturant up to its subdenaturing limit. For both proteins, the variation of effective correlation time with GdnHCl apparently shows a marginal shrink in hydrodynamic volumes under the subdenaturing condition. These results suggest that subdenaturing amounts of GdnHCl restrict the motional freedom of the internal waters, and can have considerable influence on the surface hydration. On increasing the denaturant concentration further, the dispersion amplitude drops sharply, indicating that the chaotropic action of the denaturant now runs over its own cavity water-ordering effect operative in the subdenaturing limit. The results are fundamentally important for the understanding of the susceptibility of protein structure and hydration to denaturants.