Protein Unfolded State Research Articles

Protein unfolded states populated at high and ambient pressure are similarly compact

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.

Additive Effects of Macromolecular Crowding and Confinement on Protein Stability

Folding of protein in vivo typically occurs in a solution highly crowded by macromolecules and in a confined space. It has been found that the effects of macromolecular crowding and confinement are similar in terms of the enhancement of protein stability. However, these effects are often considered separately in theoretical and simulation studies. In this study, by using coarse-grained models and Langevin dynamics, we show that the two effects are additive to each other when they are both present. Both crowding and confinement give rise to the folding temperature and the folding stability of protein. It is shown that the folding free energy change due to crowding in the confined condition can be fitted to Minton scaled particle theory by assuming a linear dependence of the effective radius of the protein unfolded state on the volume fraction of crowders.

Atomic-Level Description of Protein Folding inside the GroEL Cavity.

Chaperonins (ubiquitous facilitators of protein folding) sequester misfolded proteins within an internal cavity, thus preventing protein aggregation during the process of refolding. GroEL, a tetradecameric bacterial chaperonin, is one of the most studied chaperonins, but the role of the internal cavity in the refolding process is still unclear. It has been suggested that rather than simply isolating proteins while they refold, the GroEL cavity actively promotes protein folding. A detailed characterization of the folding dynamics and thermodynamics of protein substrates encapsulated within the cavity, however, has been difficult to obtain by experimental means, due to the system's complexity and the many steps in the folding cycle. Here, we examine the influence of the GroEL cavity on protein folding based on the results of unbiased, atomistic molecular dynamics simulations. We first verified that the computational setup, which uses a recently developed state-of-the-art force field that more accurately reproduces the aggregation propensity of unfolded states, could recapitulate the essential structural dynamics of GroEL. In these simulations, the GroEL tetradecamer was highly dynamic, transitioning among states corresponding to most of the structures that have been observed experimentally. We then simulated a small, unfolded protein both in the GroEL cavity and in bulk solution and compared the protein's folding process within these two environments. Inside the GroEL cavity, the unfolded protein interacted strongly with the disordered residues in GroEL's C-terminal tails. These interactions stabilized the protein's unfolded states relative to its compact states and increased the roughness of its folding free-energy surface, resulting in slower folding compared to the rate in solution. For larger proteins, which are more typical GroEL substrates, we speculate that these interactions may allow substrates to more quickly escape kinetic traps associated with compact, misfolded states, thereby actively promoting folding.

Dynamics of Contact Formation in Disordered Polypeptides

Molecular simulations with reliable protein and solvent models can provide information on timescales associated with reconfiguration of unfolded/disordered proteins in aqueous solution. One fundamental dynamical timescale associated with protein unfolded state that has been measured experimentally is the rate of contact formation between a probe and a quencher. We use all-atom molecular dynamics simulations to calculate the characteristic time of contact formation in disordered peptides and how it depends on peptide length. We find that our simulations with a recently proposed protein model (Amber03ws with TIP4P2005 water model) can quantitatively capture the experimental quenching times. With our simulation data, we can also address if commonly used one-dimensional (1D) diffusion models to interpret experimental data are reliable or not. We find that the results obtained from Szabo-Schulten-Schulten theory applied to a 1D diffusion model derived from the simulation data are in remarkable agreement with directly calculated rates. We also present simulation results on dynamics associated with conformational changes in the unfolded state for many long disordered proteins and compare it with the available single-molecule FRET and NMR data. Our main finding is that modern protein force fields are capable of providing accurate information on protein dynamics in the disordered states.

The unfolding effects on the protein hydration shell and partial molar volume: a computational study.

In this paper we apply the computational analysis recently proposed by our group to characterize the solvation properties of a native protein in aqueous solution, and to four model aqueous solutions of globular proteins in their unfolded states thus characterizing the protein unfolded state hydration shell and quantitatively evaluating the protein unfolded state partial molar volumes. Moreover, by using both the native and unfolded protein partial molar volumes, we obtain the corresponding variations (unfolding partial molar volumes) to be compared with the available experimental estimates. We also reconstruct the temperature and pressure dependence of the unfolding partial molar volume of Myoglobin dissecting the structural and hydration effects involved in the process.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

Unfolded-State Dynamics and Structure of Protein L Characterized by Simulation and Experiment

While several experimental techniques now exist for characterizing protein unfolded states, all-atom simulation of unfolded states has been challenging due to the long time scales and conformational sampling required. We address this problem by using a combination of accelerated calculations on graphics processor units and distributed computing to simulate tens of thousands of molecular dynamics trajectories each up to approximately 10 mus (for a total aggregate simulation time of 127 ms). We used this approach in conjunction with Trp-Cys contact quenching experiments to characterize the unfolded structure and dynamics of protein L. We employed a polymer theory method to make quantitative comparisons between high-temperature simulated and chemically denatured experimental ensembles and find that reaction-limited quenching rates calculated from simulation agree remarkably well with experiment. In both experiment and simulation, we find that unfolded-state intramolecular diffusion rates are very slow compared to highly denatured chains and that a single-residue mutation can significantly alter unfolded-state dynamics and structure. This work suggests a view of the unfolded state in which surprisingly low diffusion rates could limit folding and opens the door for all-atom molecular simulation to be a useful predictive tool for characterizing protein unfolded states along with experiments that directly measure intramolecular diffusion.

Study of Multiple Unfolding Trajectories and Unfolded States of the Protein GB1 Under the Physical Property Space

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimentional property space was reduced to a 3-dimentional principle component property space. Under the property space, the multiple unfolding trajectories look like “trees”, which have some common characters. The “root of the tree” corresponds to the native state, the “bole” homologizes the partially unfolded conformations, and the “crown” is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight “bole” and “crown” corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of “the standstill in the middle tree bole”, which may correspond to a three-state unfolding pathway. The third one has the character of “the circuitous bole” corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.

Assessment of the stability and unfolding pathways of azurin from Pseudomonas aeruginosa through the combination of denaturating osmolytes

The denaturating osmolytes urea and guanidine hydrochloride (GuHCl) interact differently with azurin from Pseudomonas aeruginosa. At room temperature, even high concentrations of urea were unable to unfold the metalloprotein: instead a “partially unfolded” intermediate state is formed. In contrast, the protein unfolded state was formed at GuHCl, concentrations above ≈3 M and the unfolded state subsequently undergoes an irreversible reaction, which was studied employing absorbance and fluorescence spectroscopic techniques. The partially unfolded protein formed in the presence of urea completely unfolds upon adding GuHCl and we report a kinetic and thermodynamic study to characterize the process, taking into consideration that the unfolded state undergoes an irreversible reaction.

Scaling Behavior and Structure of Denatured Proteins

An ensemble of random-coil conformations with no persistent structures has long been accepted as the classical model of denatured proteins due to its consistency with the experimentally determined scaling of protein sizes. However, recent NMR spectroscopy studies on proteins at high chemical denaturant concentrations suggest the presence of significant amounts of native-like structures, in contrast to the classical random-coil picture. To reconcile these seemingly controversial observations, we examine thermally denatured states of experimentally characterized proteins by using molecular dynamics simulations. For all studied proteins, we find that denatured states indeed have strong local conformational bias toward native states while a random-coil power law scaling of protein sizes is preserved. In addition, we explain why experimentally determined size of the protein creatine kinase does not follow general scaling. In simulations, we observe that this protein exhibits a stable intermediate state, the size of which is consistent with the reported experimental observation.

The structure of “unstructured” regions in peptides and proteins: Role of the polyproline II helix in protein folding and recognition*

Classical descriptions of the three-dimensional shapes of proteins usually invoke three main structures: alpha-helix, beta-sheet, and beta-turn. More recently, the polyproline II (PPII) structure has been implicated in diverse biological activities including signal transduction, transcription, cell motility, and immune response. Concurrently, evidence is accumulating that PPII structure has a significant role in the unfolded states of proteins. In this article, we connect the structural properties of PPII helices to their roles in protein recognition and protein unfolded states. The properties unique to the PPII conformation are linked to the exploitation of this structure for the molecular recognition of proteins, using peptide ligands of the Src homology 3 (SH3) domain as an example. The evidence supporting a role for PPII conformation in protein-unfolded states is also presented in the context of the forces that may stabilize the PPII conformation in unfolded polypeptides.

Earliest Events in Protein Folding: Submicrosecond Secondary Structure Formation in Reduced Cytochrome c,

Protein folding is uniquely characterized as a dynamic process by the tremendous conformational heterogeneity of the unfolded state. This conformational heterogeneity implies the possibility of significant kinetic heterogeneity in the folding dynamics, yet many folding reactions appear to proceed homogeneously, i.e., from unfolded conformations that are in equilibrium with each other and a transition state. At what point in time on the way to the folded state the kinetic heterogeneity of the unfolded state is lost to conformational equilibration has been an open question. We present evidence in this regard obtained from nanosecond far-UV optical rotatory dispersion spectroscopy of the fastest folding process observed in photoreduced cytochrome c, a secondary structure formation process proceeding on a submicrosecond time scale at high denaturant concentration (observed as a “burst” phase in millisecond stopped-flow circular dichroism studies). The kinetics of this fast folding process imply that it proceeds from a conformational ensemble that is not in equilibrium with the bulk of protein conformers during the time required to completely reduce the sample, ∼100 μs. We thus determine a lower limit on the time required for conformational equilibration of ∼10-4 s, in agreement with the previous estimate from time-resolved magnetic circular dichroism measurements on photolyzed cytochrome c-CO (Goldbeck, R. A.; Thomas, Y. G.; Chen, E.; Esquerra, R. M.; Kliger, D. S. Proc. Natl. Acad. Sci. 1999, 96, 2782−2787). Combined with an upper limit from stopped-flow measurements (Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124, 5481−5485), this allows us to bracket the conformational diffusion time, i.e., the time interval over which the kinetic heterogeneity of this protein's unfolded state is lost through conformational equilibration, within the range ∼10-4−10-3 s. This estimate of the conformational diffusion time implies that the earliest folding events, helix formation and possibly the collapse of extended conformations, proceed under an energy landscape regime wherein conformational diffusion is slow, whereas the final (milliseconds) folding phase proceeds along a classical kinetic pathway.

Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1–73, 74–108) and their noncovalent complex

We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.

Effect of hydration on thermostability

The thermostability of several serine esterases has been studied using differential scanning calorimetry. The denaturation temperature (Tm) was found to be 30–50°C higher in anhydrous environments than in aqueous solution. An almost linear decrease in Tm as a function of water activity (Aw) was observed. It is concluded that the highest productivity of an enzyme in a bioreactor would be obtained at a hydration level below optimal for catalytic activty. The data also indicates that a significant destabilisation of the protein's unfolded state occurs at low values of Aw.

Structural characterization of a protein unfolded state by solution X-ray scattering

Structural information about an unfolded state is important for the better understanding of both protein folding and protein itself. Compactness and chain statistics which are essential to characterize an unfolded state are derived by X-ray solution scattering method. In this article, the author describes the examples of studies on a protein unfolded state, such as urea denaturation of Staphylococcal nuclease, acid-denatured and molten globule states of cytochrome c, and a Staphylococcal nuclease fragment which is an unfolded state under a physiological condition. These examples suggest diversity of protein unfolded state.