Ubiquitin: a small protein folding paradigm

For the past twenty years, the small, 76-residue protein ubiquitin has been used as a model system to study protein structure, stability, folding and dynamics. In this time, ubiquitin has become a paradigm for both the experimental and computational folding communities. The folding energy landscape is now uniquely characterised with a plethora of information available on not only the native and denatured states, but partially structured states, alternatively folded states and locally unfolded states, in addition to the transition state ensemble. This Perspective focuses on the experimental characterisation of ubiquitin using a comprehensive range of biophysical techniques.     

The Energetics of a Three‐State Protein Folding System Probed by High‐Pressure Relaxation Dispersion NMR Spectroscopy

The energetic and volumetric properties of a three‐state protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure‐dependent ¹⁵N‐relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (ΔV) and isothermal compressibilities (Δκ_T) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are 100 mL mol^?1 and 40 μL mol^?1 bar^?1, respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on‐pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein. The compressibility of the intermediate is larger than that of the folded state reflecting the less rigid nature of the former relative to the latter.     

Protonation/deprotonation effects on the stability of the Trp-cage miniprotein

The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

Folding kinetics of a lattice protein via a forward flux sampling approach

We implement a forward flux sampling approach [R. J. Allen et al., J. Chem. Phys. 124, 194111 (2006)] for calculating transition rate constants and for sampling paths of protein folding events. The algorithm generates trajectories for the transition between the unfolded and folded states as chains of partially connected paths, which can be used to obtain the transition-state ensemble and the properties that characterize these intermediates. We apply this approach to Monte Carlo simulations of a model lattice protein in open space and in confined spaces of varying dimensions. We study the effect of confinement on both protein thermodynamic stability and folding kinetics; the former by mapping free-energy landscapes and the latter by the determination of rate constants and mechanistic details of the folding pathway. Our results show that, for the range of temperatures where the native state is stable, confinement of a protein destabilizes the unfolded state by reducing its entropy, resulting in increased thermodynamic stability of the folded state. Relative to the folding in open space, we find that the kinetics can be accelerated at temperatures above the temperature at which the unconfined protein folds fastest and that the rate constant increases with the number of constrained dimensions. By examining the statistical properties of the transition-state ensemble, we detect signs of a classical nucleation folding mechanism for a core of native contacts formed at an early stage of the process. This nucleus acts as folding foci and is composed of those residues that have higher probability to form native contacts in the transition-state intermediates, which can vary depending on the confinement conditions of the system.     

Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy

A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pK(a) values in unfolded protein states where chemical shift overlap is limiting. The experiment correlates side-chain carboxyl carbon chemical shifts of these residues with the backbone amide proton chemical shift of the following residue. The methodology is applied to an (15)N, (13)C labeled sample of the N-terminal SH3 domain of the Drosophila protein drk, which exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions. Residue-specific pK(a) values of side-chain carboxyl groups are presented for the first time for an unfolded protein (drk U(exch) state), determined from a pH titration. Results indicate that deviations from pK(a) values measured for model compounds are likely due to local effects, while long-range electrostatic interactions appear to be of minor importance for this protein.     

Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion

The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism. We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 degrees C. The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants. The exquisite sensitivity of the methodology to small changes in folding/unfolding rates facilitated the use of relatively low-pressure values (between 1 and 270 bar) so that pressure-induced changes to the unfolded state ensemble could be minimized. A volume change for unfolding of -81 mL/mol is measured (at 1 bar), a factor of 1.4 larger (in absolute value) than the volume difference between the transition state ensemble (TSE) and the unfolded state. Notably, the changes in the free energy difference between folded and unfolded states and in the activation free energy for folding were not linear with pressure. Thus, the difference in the isothermal compressibility upon unfolding (-0.11 mL mol(-1) bar(-1)) and, for the first time, the compressibility of the TSE relative to the unfolded state (0.15 mL mol(-1) bar(-1)) could be calculated. The results argue for a TSE that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding. The notion of a collapsed and hydrated TSE is consistent with expectations based on earlier temperature-dependent folding studies, showing that the barrier to folding at 45 degrees C is entropic (Choy, W. Y.; Zhou, Z.; Bai, Y.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 5066-5072).     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Length dependent folding kinetics of phenylacetylene oligomers: structural characterization of a kinetic trap

Using simulation to study the folding kinetics of 20-mer poly-phenylacetylene (pPA) oligomers, we find a long time scale trapped kinetic phase in the cumulative folding time distribution. This is demonstrated using molecular dynamics to simulate an ensemble of over 100 folding trajectories. The simulation data are fit to a four-state kinetic model which includes the typical folded and unfolded states, along with an intermediate state, and most surprisingly, a kinetically trapped state. Topologically diverse conformations reminiscent of alpha helices, beta turns, and sheets in proteins are observed, along with unique structures in the form of knots. The nonhelical conformations are implicated, on the basis of structural correlations to kinetic parameters, to contribute to the trapped kinetic behavior. The strong solvophobic forces which mediate the folding process and produce a stable helical folded state also serve to overstabilize the nonhelical conformations, ultimately trapping them. From our simulations, the folding time is predicted to be on the order of 2.5-12.5 mus in the presence of the trapped kinetic phase. The folding mechanism for these 20-mer chains is compared with the previously reported folding mechanism for the pPA 12-mer chains. A linear scaling relationship between the chain length and the mean first passage time is predicted in the absence of the trapped kinetic phase. We discuss the major implications of this discovery in the design of self-assembling nanostructures.     

The Trp cage: folding kinetics and unfolded state topology via molecular dynamics simulations

Using over 75 mus of molecular dynamics simulation, we have generated several thousand folding simulations of the 20-residue Trp cage at experimental temperature and solvent viscosity. A total of 116 independent folding simulations reach RMSDcalpha values below 3 A RMSDcalpha, some as close as 1.4 A RMSDcalpha. We estimate a folding time of 5.5+/-3.5 mus, a rate that is in reasonable agreement with experimental kinetics. Finally, we characterize both the folded and unfolded ensemble under native conditions and note that the average topology of the unfolded ensemble is very similar to the topology of the native state.     

Estimation of protein folding rate from Monte Carlo simulations and entropy capacity

The problem of protein self-organization is one of the most important problems of molecular biology nowadays. Despite the recent success in the understanding of general principles of protein folding, details of this process are yet to be elucidated. Moreover, the prediction of protein folding rates has its own practical value due to the fact that aggregation directly depends on the rate of protein folding. The time of folding has been calculated for 67 proteins with known experimental data at the point of thermodynamic equilibrium between unfolded and native states using a Monte Carlo model where each residue is considered to be either folded as in the native state or completely disordered. The times of folding for 67 proteins which reach the native state within the limit of 10(8) Monte Carlo steps are in a good correlation with the experimentally measured folding rate at the mid-transition point (the correlation coefficient is -0.82). Theoretical consideration of a capillarity model for the process of protein folding demonstrates that the difference in the folding rate for proteins sharing more spherical and less spherical folds is the result of differences in the conformational entropy due to a larger surface of the boundary between folded and unfolded phases in the transition state for proteins with more spherical fold. The capillarity model allows us to predict the folding rate at the same level of correlation as by Monte Carlo simulations. The calculated model entropy capacity (conformational entropy per residue divided by the average contact energy per residue) for 67 proteins correlates by about 78% with the experimentally measured folding rate at the mid-transition point.     

Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: an application to the folding of a Fyn SH3 domain mutant

The three-site exchange folding reaction of an (15)N-labeled, highly deuterated Gly48Met mutant of the Fyn SH3 domain has been characterized at 25 degrees C using a suite of six CPMG-type relaxation dispersion experiments that measure exchange contributions to backbone (1)H and (15)N transverse relaxation rates in proteins. It is shown that this suite of experiments allows the extraction of all the parameters of this multisite exchange process in a robust manner, including chemical shift differences between exchanging states, from a data set recorded at only a single temperature. The populations of the exchanging folded, intermediate, and unfolded states that are fit are 94, 0.7, and 5%, respectively. Despite the small fraction of the intermediate, structural information is obtained for this state that is consistent with the picture of SH3 domain folding that has emerged from other studies. Taken together, the six dispersion experiments facilitate the complete reconstruction of (1)H-(15)N correlation spectra for the unfolded and intermediate states that are "invisible" in even the most sensitive of NMR experiments.     

Direct measurement of tertiary contact cooperativity in RNA folding

All structured biological macromolecules must overcome the thermodynamic folding problem to populate a unique functional state among a vast ensemble of unfolded and alternate conformations. The exploration of cooperativity in protein folding has helped reveal and distinguish the underlying mechanistic solutions to this folding problem. Analogous dissections of RNA tertiary stability remain elusive, however, despite the central biological importance of folded RNA molecules and the potential to reveal fundamental properties of structured macromolecules via comparisons of protein and RNA folding. We report a direct quantitative measure of tertiary contact cooperativity in a folded RNA. We precisely measured the stability of an independently folding P4-P6 domain from the Tetrahymena thermophila group I intron by single molecule fluorescence resonance energy transfer (smFRET). Using wild-type and mutant RNAs, we found that cooperativity between the two tertiary contacts enhances P4-P6 stability by 3.2 +/- 0.2 kcal/mol.     

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.     

On the application of CZE to the study of protein denaturation

Experimental mobilities obtained from CZE are used to study protein denaturation through a model based on known physicochemical theories. This model is able to provide additional information concerning the folded and unfolded protein states from mobility data. Its use comprises first the evaluation of relevant parameters of the protein microstates like the electrostatic free energy, apart from the classical conformational free energy, and second the expression of raw experimental data concerning the folding-unfolding transition into more specific physicochemical parameters like protein hydrodynamic radius, net charge number, and hydration. Spurious effects that are intrinsic to the experimental evaluation of the mobility of protein states, like BGE viscosity, pH, and ionic strength variations accompanying the changes of the denaturant agent intensity are eliminated. In order to illustrate the proposal of this work, two case studies are considered here. The first one concerns thermal and urea denaturations of horse heart ferricytochrome c and the second one involves thermal denaturation of hen egg-white lysozyme. Thus, relevant theoretical thermodynamic considerations of the folded-unfolded protein transition are presented, where the electrostatic free energy is included explicitly in the effective free energy. It is found that this transition involves sharp increases of hydrodynamic radius and protein hydration.     

Probing electrostatic interactions and structural changes in highly charged protein polyanions by conformer-selective photoelectron spectroscopy

We have recorded the first conformer-selective photoelectron spectra of a protein polyanion in the gas-phase. Bovine cytochrome c protein was studied in 8 different negative charge states ranging from 5- to 12-. Electron binding energies were extracted for all charge states and used as a direct probe of intramolecular Coulomb repulsion. Comparison of experimental results with simulations shows that the experimental outcome can be reproduced with a simple electrostatic model. Energetics are consistent with a structural transition from a folded to an unfolded conformational state of the protein as the number of charges increases. Furthermore, the additional ion-mobility data show that the onset of unfolding can be assigned to charge state 6- where three conformers can be distinguished.     

New scenarios of protein folding can occur on the ribosome

Identifying and understanding the differences between protein folding in bulk solution and in the cell is a crucial challenge facing biology. Using Langevin dynamics, we have simulated intact ribosomes containing five different nascent chains arrested at different stages of their synthesis such that each nascent chain can fold and unfold at or near the exit tunnel vestibule. We find that the native state is destabilized close to the ribosome surface due to an increase in unfolded state entropy and a decrease in native state entropy; the former arises because the unfolded ensemble tends to behave as an expanded random coil near the ribosome and a semicompact globule in bulk solution. In addition, the unfolded ensemble of the nascent chain adopts a highly anisotropic shape near the ribosome surface and the cooperativity of the folding-unfolding transition is decreased due to the appearance of partially folded structures that are not populated in bulk solution. The results show, in light of these effects, that with increasing nascent chain length folding rates increase in a linear manner and unfolding rates decrease, with larger and topologically more complex folds being the most highly perturbed by the ribosome. Analysis of folding trajectories, initiated by temperature quench, reveals the transition state ensemble is driven toward compaction and greater native-like structure by interactions with the ribosome surface and exit vestibule. Furthermore, the diversity of folding pathways decreases and the probability increases of initiating folding via the N-terminus on the ribosome. We show that all of these findings are equally applicable to the situation in which protein folding occurs during continuous (non-arrested) translation provided that the time scales of folding and unfolding are much faster than the time scale of monomer addition to the growing nascent chain, which results in a quasi-equilibrium process. These substantial ribosome-induced perturbations to almost all aspects of protein folding indicate that folding scenarios that are distinct from those of bulk solution can occur on the ribosome.     

Contribution of charged groups to the enthalpic stabilization of the folded states of globular proteins

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO(-)) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while N(+)(3) groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously.     

Direct test of the Gaussian-chain model for treating residual charge-charge interactions in the unfolded state of proteins

The Gaussian-chain model for treating residual charge-charge interactions was critically tested by recent experimental pK(a) results for individual Asp, Glu, and His residues in the unfolded drkN SH3 domain. Predicted pK(a)'s were in good agreement with experiment. The clustering of Asp and Glu residues along the sequence was suggested to limit pK(a) shifts and contribute to the folding stability by destabilizing the unfolded state.     

Electrostatics of nucleic acid folding under conformational constraint

RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg(2+) stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson-Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg(2+) titrations. This finding is consistent with previous work, suggesting that Poisson-Boltzmann and other mean-field theories fail for higher valency cations where ion-ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.