Exploring the Cooperativity of the Fast Folding Reaction of a Small Protein Using Pulsed Thiol Labeling and Mass Spectrometry

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To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.noncooperative ͉ submillisecond protein folding

Section: Absence Of a Significant Free Energy Barrier During The Sub-msmentioning

confidence: 85%

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Sinha

Udgaonkar

2008

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“…1A). We used two different thiol-specific labeling reagents, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and methyl metahnethiosulfonate (MMTS), both of which react rapidly with exposed thiolate groups (33)(34)(35), to measure the changes in solvent accessibility during unfolding ( Fig. 5 A−C).…”

Section: Initial Expansion Is Not Accompanied By Disruption Of Secondarymentioning

confidence: 99%

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

Jha

Marqusee

2014

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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).protein unfolding | dry molten globule | steady-state FRET D enaturants such as guanidinium chloride (GdmCl) and urea are classic perturbants used to probe the thermodynamics and kinetics of protein conformational changes, although their mechanism of action is poorly understood (1-15). In some of these studies, a dry molten globular (DMG) state has been observed on the native side of the free-energy barrier (16-18). The DMG is an expanded form of the native protein in which at least some of the side-chain packing interactions are disrupted without solvation of the hydrophobic core; it was originally postulated to explain heat-induced unfolding of proteins (19,20). Recently, unfolding intermediates resembling the DMG have also been observed in the absence of denaturants, and it has been suggested that the DMG exists in rapid equilibrium with the native state (21-23). The fact that the DMG can be observed by the addition of denaturants is, however, counterintuitive, because traditional models of chemical denaturation rely on changes in solvent-accessible surface area (SASA) (3,24,25) and there is no notable change in SASA during the formation of the DMG.Recent computational studies have suggested an alternative model of chemical denaturation in which denaturants first interact directly with the protein surface, causing the protein to swell, and then penetrate the core (the so-called "direct interaction" mechanism) (9,11,12,26). One of the consequences of this model is that denaturants unfold proteins in two steps (9, 12). In the first step, denaturant molecules displace water molecules within the fir...

“…Amide proton exchange is used most commonly to monitor such differential reactivity (5-9), but its widespread use to assess biological function typically is limited by the need for specialized instrumentation and relatively large amounts of protein. Recently, cysteine reactivity (10)(11)(12)(13)(14) and proteolysis (15) have emerged as alternative means to determine rates of protein (un)folding and estimate protein stabilities. Here we present a method, quantitative cysteine reactivity (QCR), in which protein stability is determined by monitoring the reactivity of cysteine residues buried in the hydrophobic core of proteins.…”

mentioning

confidence: 99%

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Isom

Vardy

Oas

et al. 2010

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCRderived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics. Macromolecular stability is therefore one of the most fundamental thermodynamic measures in biochemistry by quantitatively reporting on structure-function relationships to provide a universal monitor for biochemical function.There are two distinct approaches for determining protein stability (4). The first measures the free energy of protein (un)folding under equilibrium conditions by assessing the fraction of the native state using spectroscopy, hydrodynamic observations, functional assays, or calorimetry. The second exploits the relationship between protein dynamics and stability by monitoring the differential reactivity of internal chemical groups in native and unfolded states. This second approach measures conformational free energies, which under appropriate conditions corresponds to global protein stability. Amide proton exchange is used most commonly to monitor such differential reactivity (5-9), but its widespread use to assess biological function typically is limited by the need for specialized instrumentation and relatively large amounts of protein. Recently, cysteine reactivity (10-14) and proteolysis (15) have emerged as alternative means to determine rates of protein (un)folding and estimate protein stabilities. Here we present a method, quantitative cysteine reactivity (QCR), in which protein stability is determined by monitoring the reactivity of cysteine residues buried in the hydrophobic core of proteins. This approach has the advantage over more traditional methods for measuring protein stability in that it requires only picomoles (nanograms) of protein, uses simple instrumentation accessible to any lab, can be reasonably high throughput, and can provide site-specific thermodynamic information. QCR can be used to determine apparent protein stabilities rapidly and accurately, construct Gibbs-Helmholtz stability profiles, measure ligand binding over a large range of ligand concentrations and affinities, and infer e...