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Admitting the “Native”, “Unfolded” and “Fibril” states as the three basic generic states of proteins in nature, each of which is characterized with its partial molar volume, here we predict that the interconversion among these generic states N, U, F may be performed simply by making a temporal excursion into the so called “the high-pressure regime”, created artificially by putting the system under sufficiently high hydrostatic pressure, where we convert N to U and F to U, and then back to “the low-pressure regime” (the “Anfinsen regime”), where we convert U back to N (U→N). Provided that the solution conditions (temperature, pH, etc.) remain largely the same, the idea provides a general method for choosing N, U, or F of a protein, to a great extent at will, assisted by the proper use of the external perturbation pressure. A successful experiment is demonstrated for the case of hen lysozyme, for which the amyloid fibril state F prepared at 1 bar is turned almost fully back into its original native state N at 1 bar by going through the “the high-pressure regime”. The outstanding simplicity and effectiveness of pressure in controlling the conformational state of a protein are expected to have a wide variety of applications both in basic and applied bioscience in the future.

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis , as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-molecule Förster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been observed for many proteins at low denaturant concentration, IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an additional collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures

Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute–protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute–protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, 〈r −6〉norm, and an effective correlation time, τc. The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute–protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the 〈r −6〉norm values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are “uncoupled” (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes.

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal·Å−3.