Santosh Kumar Jha

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.

Continuous dissolution of structure during the unfolding of a small protein

The unfolding kinetics of many small proteins appears to be first order, when measured by ensemble-averaging probes such as fluorescence and circular dichroism. For one such protein, monellin, it is shown here that hidden behind this deceptive simplicity is a complexity that becomes evident with the use of experimental probes that are able to discriminate between different conformations in an ensemble of structures. In this study, the unfolding of monellin has been probed by measurement of the changes in the distributions of 4 different intramolecular distances, using a multisite, time-resolved fluorescence resonance energy transfer methodology. During the course of unfolding, the protein molecules are seen to undergo slow and continuous, diffusive swelling. The swelling process can be modeled as the slow diffusive swelling of a Rouse-like chain with some additional noncovalent, intramolecular interactions. Here, we show that specific structure is lost during the swelling process gradually, and not in an all-or-none manner, during unfolding.

Exploring the cooperativity of the fast folding reaction of a small protein using pulsed thiol labeling and mass spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

Significance Guanidinium chloride (GdmCl) has been used to modulate the stability of proteins for more than 50 years; surprisingly, however, the molecular mechanism of its action is still poorly understood. Here, we provide direct kinetic evidence for the hypothesis that GdmCl unfolds proteins by a two-step mechanism. In the first step, it binds to the protein surface, resulting in the formation of a “dry molten globule,” an expanded form of the native protein with a dry core. Core solvation and global structural disruption occur in the second step. The observation of a dry molten globule during unfolding indicates that dispersion forces, and not only the hydrophobic effect, also play an important role in stabilizing proteins. Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry.

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

Characterization of deamidation of barstar using electrospray ionization quadrupole time-of-flight mass spectrometry, which stabilizes an equilibrium unfolding intermediate.

Deamidation of asparaginyl residues is a common posttranslational modification in proteins and has been studied extensively because of its important biological effects, such as those on enzymatic activity, protein folding, and proteolytic degradation. However, characterization of the sites of deamidation of a protein has been a difficult analytical problem. In this study, mass spectrometry has been used as an analytical tool to characterize the deamidation of barstar, an RNAse inhibitor. Upon incubation of the protein at alkaline pH for 5 h, intact mass analysis of barstar, using electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI QToF MS), indicated an increase in the mass of +2 Da, suggesting possible deamidation of the protein. The sites of deamidation have been identified using the conventional bottom‐up approach using a capillary liquid chromatography connected on line to an ESI QToF mass spectrometer and top down approach by direct infusion of the intact protein and fragmenting inside MS. These chemical modifications are shown to lead to stabilization of an unfolding intermediate, which can be observed in equilibrium unfolding studies.

An Alternatively Packed Dry Molten Globule-like Intermediate in the Native State Ensemble of a Multidomain Protein

It has been difficult to quantify the degree of side-chain conformational heterogeneity in the native (N) state ensemble of proteins and the relative energetic contributions of the side-chain packing and the hydrophobic effect in protein stability. Here, we show using multiple site-specific spectroscopic probes and tools of thermodynamics that the N state ensemble of a multidomain protein contains an equilibrium intermediate (I) whose interdomain region resembles a dry molten globule. In the I state, a tryptophan residue in the interdomain region is alternatively packed, but its secondary structure and intradomain packing are N-like. The I state also has a larger interdomain distance, but the domain-domain interface is dry and molten. Our results indicate that hydrophobic desolvation and side-chain packing are decoupled during protein folding and that interdomain packing interactions have an important energetic contribution in protein stability. Dynamic interconversion between alternatively packed N-like states could be important for multiple allosteric and ligand binding functions of this protein.