High Denaturant Concentrations Research Articles

SAXS versus FRET: A Matter of Heterogeneity?

Determining how conformational properties of unfolded proteins vary as a function of denaturant concentration has important implications for understanding the early stages of protein folding, and for our understanding of protein-solvent interactions. Upon dilution from a high concentration of denaturant into native conditions, does an unfolded protein undergo collapse first followed by folding, or does the chain remain expanded before folding and collapse occur concomitantly? The global dimensions of a protein are captured by its radius of gyration (Rg), meaning that, in principle, this question should be simple to answer by measuring Rg as a function of denaturant concentration.

Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions.

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.

Evidence for the residual tertiary structure in the urea-unfolded form of bacteriophage T5 endolysin

Using high-resolution NMR spectroscopy, we studied peculiarities of the unfolding process of the bacteriophage T5 endolysin (EndoT5) by strong denaturants. It was shown that in the absence of zinc ions this protein is mostly unfolded in the solution of 8 M urea or 6 M guanidine hydrochloride. However, in the presence of zinc ions EndoT5 unfolding can be achieved only in acidic solutions (at pH < 4.0), whereas at pH > 4.0 NMR spectra of the metal-bound protein (Zn2+–Ca2+–EndoT5 or Zn2+–EndoT5 complexes) exhibit a few chemical shifts characteristic of the native or native-like proteins. Our data, including the pH–titration curve with the pK of ~5, suggested involvement of the zinc-binding histidines in the stabilization of this protein. Up-field signals that appear in the NMR spectra of apo-EndoT5 in the presence of high concentrations of strong denaturants are probably derived from the amino acid residues included in the formation of structured hydrophobic cluster, which likely corresponds to the 81–93 region of EndoT5 and contains some residual tertiary structure. It is possible also that this hydrophobic fragment serves as a foundation for the formation of structured cluster in the unfolded state.

Conformations of a Metastable SH3 Domain Characterized by smFRET and an Excluded-Volume Polymer Model

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.

Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype.

Phenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrP(Sc) conformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was ∼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrP(Sc) polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrP(Sc) differed for the CWD strains in a host-dependent manner. WST CWD produced PrP(Sc) amyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrP(Sc) from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrP(Sc) properties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent of Prnp can alter the PrP(Sc) conformation of certain prion strains, leading to changes in the biophysical properties of PrP(Sc), neuropathology, and clinical prion disease. Prions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

Impact of confinement on proteins concentrated in lithocholic acid based organic nanotubes

Organic nanotubes form in aqueous solution near physiological pH by self-assembly of lithocholic acid (LCA) with inner diameters of 20–40nm. The encapsulation of enhanced green fluorescent protein (eGFP) and resultant confinement effect for eGFP within these nanotubes is studied via confocal microscopy. Timed release rate studies of eGFP encapsulated in LCA nanotubes and fluorescence recovery after photobleaching (FRAP) indicate that the diffusive transport of eGFP out of and/or within the nanotubes is very slow, in contrast to the rapid introduction of eGFP into the nanotubes. By encapsulating two fluorescent proteins in LCA nanotubes, eGFP and mCherry, as a fluorescence resonance energy transfer (FRET) pair, the FRET efficiencies are determined using FRET imaging microscopy at three different protein concentrations with a fixed donor-to-acceptor ratio of 1:1. Förster theory reveals that the proteins are spatially separated by 4.8–7.2nm in distance inside these nanotubes. The biomimetic nanochannels of LCA nanotubes not only afford a confining effect on eGFP that results in enhanced chemical and thermal stability under conditions of high denaturant concentration and temperature, but also function as protein concentrators for enriching protein in the nanochannels from a diluted protein solution by up to two orders of magnitude.

A small deletion in C9orf72 hides a proportion of expansion carriers in FTLD

Frontotemporal lobar degeneration is a highly familial disease and the most common known genetic cause is the repeat expansion mutation in the gene C9orf72. We have identified 2 brothers with an expansion mutation in C9orf72 using Southern blotting that is undetectable using repeat-primed polymerase chain reaction. Sequencing using high concentrations of DNA denaturants of a bacterial artificial chromosome clone obtained from one of the brothers identified a 10-base pair deletion adjacent to the expansion that presumably confers strong secondary structure that interferes with the genotyping. Using an alternative method, we have identified missed expansion carriers in our cohort, and this number has increased by approximately 25%. This observation has important implications for patients undergoing genetic testing for C9orf72.

Targeting chemical and thermal stability of ovalbumin by simulated honey sugar cocktail

Effect of simulated honey sugar cocktail (SHSC) on chemical and thermal stability of ovalbumin (OVA) was investigated using multiple-spectroscopic techniques. Urea-induced denaturation of OVA produced a transition, characterized by the start-, the mid- and the end-points at 3.2M, 5.9/5.6M and 8.5/8.0M urea, respectively, when studied by MRE222nm and tryptophan fluorescence measurements. Presence of 10% or 20% (w/v) SHSC in the incubation mixture shifted the transition curve towards higher urea concentration in a concentration dependent manner. A comparison of far- and near-UV CD, UV-difference, ANS fluorescence and 3-D fluorescence spectral results of native OVA and 5.9M urea-denatured OVA (U-OVA), obtained in the absence and the presence of 20% (w/v) SHSC suggested SHSC-induced stabilization of U-OVA. Furthermore, a significant shift towards higher denaturant concentration was also noticed in the GdnHCl and thermal transition curves of OVA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested stabilization of OVA against chemical and thermal denaturations by SHSC.

Towards increasing chemical and thermal stability of lysozyme with a simulated honey sugar cocktail

Guanidine hydrochloride (GdnHCl) and thermal denaturation studies on lysozyme were made in the absence and the presence of a simulated honey sugar cocktail (SHSC). GdnHCl denaturation curve was shifted towards higher denaturant concentration along with an increase in value in the presence of SHSC, when studied by MRE222 nm and tryptophan fluorescence measurements. Far- and near-UV CD as well as ANS fluorescence spectra of the partially-denatured lysozyme indicated greater retention of the folded structure in the presence of SHSC. The thermal transition curve showed a shift towards the higher temperature range along with an increase in the melting temperature in the presence of SHSC, when studied by MRE222 nm and DSC measurements. Taken together, all these results suggested SHSC-induced chemical and thermal stabilization of lysozyme.

High stability and cooperative unfolding of α-synuclein oligomers.

Many neurodegenerative diseases are linked with formation of amyloid aggregates. It is increasingly accepted that not the fibrils but rather oligomeric species are responsible for degeneration of neuronal cells. Strong evidence suggests that in Parkinson's disease (PD), cytotoxic α-synuclein (αSN) oligomers are key to pathogenicity. Nevertheless, insight into the oligomers' molecular properties remains scarce. Here we show that αSN oligomers, despite a large amount of disordered structure, are remarkably stable against extreme pH, temperature, and even molar amounts of chemical denaturants, though they undergo cooperative unfolding at higher denaturant concentrations. Mutants found in familial PD lead to slightly larger oligomers whose stabilities are very similar to that of wild-type αSN. Isolated oligomers do not revert to monomers but predominantly form larger aggregates consisting of stacked oligomers, suggesting that they are off-pathway relative to the process of fibril formation. We also demonstrate that 4-(dicyanovinyl)julolidine (DCVJ) can be used as a specific probe for detection of αSN oligomers. The high stability of the αSN oligomer indicates that therapeutic strategies should aim to prevent the formation of or passivate rather than dissociate this cytotoxic species.

Factor defining the effects of glycine betaine on the thermodynamic stability and internal dynamics of horse cytochrome C.

A compatible osmolyte such as glycine betaine (GB) and low concentrations of a denaturant constrain the internal dynamics of natively folded carbonmonoxycytochrome c (NCO) at pH 7.0. GB and subdenaturing concentrations of guanidine hydrochloride (GdnHCl) or urea have a cumulative effect on the constrained dynamics of NCO. At higher denaturant concentrations, large-scale unfolding fluctuations dominate the dynamics and inclusion of GB opposes the structural fluctuations that cause unfolding of the protein. These deductions are made from kinetic and thermodynamic parameters measured for the CO dissociation reaction of NCO at varying concentrations of denaturant in the absence and presence of 1.0 M GB. Intermolecular docking between horse ferrocytochrome c and a denaturant or GB reveals that the denaturant-mediated constrained dynamics of the protein is due to polyfunctional interactions between the denaturant and different groups of protein while the GB-mediated restricted dynamics of the protein arises from both the direct interactions of GB with different side chains of Lys or Arg residues of the protein and indirect interactions of GB with the protein surface. Thermodynamic analysis of the thermal and GdnHCl-induced unfolding curves of ferrocytochrome c measured in the absence and presence of 1.0 M GB at pH 7.0 indicates that GB increases the thermodynamic stability of ferrocytochrome c at neutral pH. Analysis of thermal and urea-induced unfolding curves of ferricytochrome c measured at different GdnHCl concentrations in the absence and presence of 0.5-1.0 M GB at pH 7.0 and 3.8 suggests that GB counteracts the destabilizing effect of the denaturant at pH 7.0 but exhibits an additive effect on the destabilizing effect of the denaturant at pH 3.8.

High-Energy Intermediates in Protein Unfolding Characterized by Thiol Labeling under Nativelike Conditions

A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.

Stabilizing effect of small concentrations of PAMAM dendrimers at the insulin aggregation

Dendrimers’ action on proteins and peptides has a dual and controversial character. On one hand, they dissolve prion protein and amyloid fibrils aggregates, which are otherwise only soluble in solvents containing both detergents and high denaturant concentrations. On the other hand they are able to destabilize proteins in generation dependent manner. In present work we estimated the influence of small concentrations (up to 1.4μg/ml) of cationic, neutral and anionic poly(amidoamine) dendrimers of 3rd and 4th generations on dithiotreitol induced aggregation of insulin. It was found that cationic dendrimers decreased the insulin aggregation, while anionic and neutral ones did not. At the same time, destabilizing effect of dendrimers on insulin structure was not observed. The conclusion was made that small concentrations of dendrimers can be applied to prevent or decrease the formation of misfolded structures of protein.

Critical Evaluation of the Two-State Model Describing the Equilibrium Unfolding of the PI3K SH3 Domain by Time-Resolved Fluorescence Resonance Energy Transfer

It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model.

DNA loop domain organization as revealed by single-cell gel electrophoresis

At higher order levels chromatin is organized into loops. This looping, which plays an important role in transcription regulation and other processes, remains poorly understood. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay). The migration of a part of the loops was shown to be reversible after switching off electrophoresis and to be sensitive to intercalation-induced changes in supercoiling. Another group of the loops migrates rapidly, the rate being insensitive to the supercoiling level. The largest part of the loops cannot migrate at all, presumably because of their large size. The loop ends can be detached in the presence of high concentrations of intercalators or protein denaturants, thus increasing the fraction of DNA that cannot migrate in the gel. The distribution of the loop length up to 100kilobases appears to be consistent with the fractal globule organization.

N-Propanol Based Solubilization Buffer Enhances Refolding Yield of Inclusion Body Protein by Populating Intermediates to the Folding Pathway

Most of the times, high level expression of recombinant proteins in bacteria results in accumulation of recombinant proteins into inclusion body (IB) aggregates. To obtain native protein from these aggregates, it is necessary to solubilize these aggregates followed by refolding of solubilized protein by appropriate refolding method. Conventionally, high concentration of denaturant like urea or guanidinium chloride (GdmCl) is used for solubilization of inclusion bodies which often results into aggregation of protein during refolding process. In the present study we have evaluated a novel solubilization method using n-propanol in presence of low concentration of urea. n-Propanol based solubilization agent was compared with traditional solubilization agents like 8 M urea and 6 M GdmCl for solubilization efficiency, structure and stability of the solubilized model protein, recombinant human growth hormone (hGH). hGH IBs were found to be tough and were only solubilized efficiently in presence of high concentration of denaturants (8 M urea or 6 M GdmCl). 4 to 6 M n-propanol in combination with 2 M urea was sufficient for the efficient solubilization of hGH IBs. Aggregation during refolding was also studied and it was found that solubilization with n-propanol based buffer resulted into bioactive hGH without aggregation giving significantly higher refolding yield in comparison to those obtained with urea and GdmCl based buffers which resulted in aggregation of hGH during refolding. From the results obtained, it can be concluded that solubilization of hGH IBs in n-propanol based buffer results in a partially folded folding intermediate of hGH which readily folds into native form on dilution with reduced chances of protein getting into aggregation pathway.

The Intrinsically Unstable SH3-DRKN Protein: Compactness, Conformations and Speed

SH3 is a common binding domain that mediates protein-protein interactions. The N-terminal SH3 domain of the Drosophila adaptor protein Drk is marginally stable under physiological conditions, showing nearly 50/50 equilibrium between the folded and unfolded states. Due to the dynamic ensemble nature of this unfolded state under non-denaturing conditions, the structural characterization of SH3 is difficult using ensemble measurements. Experimental data have been used to define sets of heterogeneous conformations but ensemble averaging minimizes the information content [1]. Importantly, little is known about interconversion rates within the unfolded ensemble.We performed fluorescence correlation spectroscopy (FCS) experiments on the wild-type and on a single-site mutant stabilizing the folded state. Based on this data, we estimated the size (hydrodynamic radii), of the folded (Rf), unfolded (Ru) and denatured (Rd) states. The size of unfolded state measured by FCS is in excellent agreement with the NMR-measured value, Ru/Rf = 1.30 ± 0.01 [1]. Quite surprisingly, our results indicate that the unfolded state under non-denaturing conditions is less compact than the chemically-denatured state, as Rd/Rf = 1.21 ± 0.03. This agrees with previously reported NMR and SAXS data when adequate shape factors for different conformations are considered. Using an environment sensitive dye and fitting the full correlation curve reveals a fast (∼200 ns) process within the unfolded ensemble that is absent in the folded state.Single-molecule FRET experiments were performed to quantify end-to-end distance distributions. We probed directly the conformational heterogeneity of the wild-type protein as well as the response to denaturants and osmolytes. Quite surprisingly, N- and the C-termini remained in close proximity at high denaturant concentrations for ca. 40% of the conformations, suggesting that DrkN-SH3behaves at least partially like a disordered circular chain.Reference:1. J.A. Marsh and J.D. Forman-Kay, Proteins 80(2):556-572 (2012)

Theory of the protein equilibrium population snapshot by H/D exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS) method used to obtain protein folding energies/rates and selected supporting experimental evidence

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a “snapshot” of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔGH2O. In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.