Denatured-state ensemble and the early-stage folding of the G29A mutant of the B-domain of protein A

The folding mechanism of the G29A mutant of the B-domain of protein A (BdpA) has been studied by all-atom molecular dynamics simulation using AMBER force field (ff03) and generalized Born continuum solvent model. Started from the extended chain conformation, a total of 16 simulations (400 ns each) at 300 K captured some early folding events of the G29A mutant of BdpA. In one of the 16 trajectories, the G29A mutant folded within 2.8 A (root mean square) of the wild-type NMR structure. We observed that the fast burial of hydrophobic residues was the driving force to bring the distant residues into close proximity. The initiation of the helix I and III occurred during the stage of hydrophobic collapse. The initiation and growth of the helix II was slow. Both the secondary structure formation and the development of the native tertiary contacts suggested a multistage folding process. Clustering analysis indicated that two helix species (helices I and III) could be intermediates. Further analysis revealed that the hydrophobic residues of partially folded helix II formed nativelike hydrophobic contacts with helices I and III that stabilized a nativelike state and delayed the completion of folding of the entire protein. The details of the early folding process were compared with other theoretical and experimental studies. It was found that a nativelike hydrophobic cluster was formed by residues including F(30), I(31), L(34), L(44), L(45), and A(48) that prevented further development of the native structures, and breaking the hydrophobic cluster like this one contributed to the rate-limiting step. This was in complete agreement with the recent kinetic measurements in which mutations of these residues to Gly and Ala substantially increased the folding rates by as much as 60 times. Apparently, destabilization of nonnative states dramatically enhanced the folding rates.     

Ab initio folding of albumin binding domain from all-atom molecular dynamics simulation

Ab initio folding with all-atom model remains to be a difficult task even for small proteins. In this report, we conducted an accumulated 24 mus simulations on the wild type and two mutants of albumin binding domain (ABD) using the AMBER FF03 all-atom force field and a generalized-Born solvation model. Folding events have been observed in multiple trajectories, and the best folded structures achieved root-mean-square deviation (RMSD) of 2.0 A. The folding of this three-helix bundle protein followed a diffusion-collision process, where substantial formation of the individual helices was critical and preceded the global packing. Owing to the difference in the intrinsic helicity, helix I formed faster than the other two helices. The order of the formation of helices II and III varied in different trajectories, indicating heterogeneity of the folding process. The slightly shifted boundaries of the helical segments had direct impact on the global packing, suggesting room for improvement on the simulation force field and solvation model.     

Folding processes of the B domain of protein A to the native state observed in all-atom ab initio folding simulations

Reaching the native states of small proteins, a necessary step towards a comprehensive understanding of the folding mechanisms, has remained a tremendous challenge to ab initio protein folding simulations despite the extensive effort. In this work, the folding process of the B domain of protein A (BdpA) has been simulated by both conventional and replica exchange molecular dynamics using AMBER FF03 all-atom force field. Started from an extended chain, a total of 40 conventional (each to 1.0 micros) and two sets of replica exchange (each to 200.0 ns per replica) molecular dynamics simulations were performed with different generalized-Born solvation models and temperature control schemes. The improvements in both the force field and solvent model allowed successful simulations of the folding process to the native state as demonstrated by the 0.80 A C(alpha) root mean square deviation (RMSD) of the best folded structure. The most populated conformation was the native folded structure with a high population. This was a significant improvement over the 2.8 A C(alpha) RMSD of the best nativelike structures from previous ab initio folding studies on BdpA. To the best of our knowledge, our results demonstrate, for the first time, that ab initio simulations can reach the native state of BdpA. Consistent with experimental observations, including Phi-value analyses, formation of helix II/III hairpin was a crucial step that provides a template upon which helix I could form and the folding process could complete. Early formation of helix III was observed which is consistent with the experimental results of higher residual helical content of isolated helix III among the three helices. The calculated temperature-dependent profile and the melting temperature were in close agreement with the experimental results. The simulations further revealed that phenylalanine 31 may play critical to achieve the correct packing of the three helices which is consistent with the experimental observation. In addition to the mechanistic studies, an ab initio structure prediction was also conducted based on both the physical energy and a statistical potential. Based on the lowest physical energy, the predicted structure was 2.0 A C(alpha) RMSD away from the experimentally determined structure.     

Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry

The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.     

Measuring pK(a) values in protein folding transition state ensembles by NMR spectroscopy

Protein folding kinetic data have been obtained for the marginally stable N-terminal SH3 domain of the Drosophila protein drk as a function of pH in order to investigate the electrostatic properties of Asp8 in the folding transition state ensemble. The slow exchange between folded and unfolded forms of the protein gives rise to separate NMR resonances for both folded and unfolded states at equilibrium. As a result, kinetic data can be derived from magnetization transfer between these two states without the need for denaturants. Using the fact that ionization of Asp8 dominates the electrostatic behavior of the protein between pH 2 and 3, along with pKa values for titrating groups in both folded and unfolded states that have been determined in a previous study, values of 2.9 +/- 0.1 and 3.3 +/- 0.2 are obtained for the pKa of Asp8 in the transition state for the wild-type protein and for a His7Ala mutant, respectively. The data are consistent with the partial formation in the transition state ensemble of an Asp8 side chain carboxylate-a Lys21 backbone amide interaction that represents a highly conserved contact in folded SH3 domains.     

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.     

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.     

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.     

A lattice protein with an amyloidogenic latent state: stability and folding kinetics

We have designed a model lattice protein that has two stable folded states, the lower free energy native state and a latent state of somewhat higher energy. The two states have a sizable part of their structures in common (two "alpha-helices") and differ in the content of "alpha-helices" and "beta-strands" in the rest of their structures; i.e. for the native state, this part is alpha-helical, and for the latent state it is composed of beta-strands. Thus, the lattice protein free energy surface mimics that of amyloidogenic proteins that form well organized fibrils under appropriate conditions. A Go-like potential was used and the folding process was simulated with a Monte Carlo method. To gain insight into the equilibrium free energy surface and the folding kinetics, we have combined standard approaches (reduced free energy surfaces, contact maps, time-dependent populations of the characteristic states, and folding time distributions) with a new approach. The latter is based on a principal coordinate analysis of the entire set of contacts, which makes possible the introduction of unbiased reaction coordinates and the construction of a kinetic network for the folding process. The system is found to have four characteristic basins, namely a semicompact globule, an on-pathway intermediate (the bifurcation basin), and the native and latent states. The bifurcation basin is shallow and consists of the structure common to the native and latent states, with the rest disorganized. On the basis of the simulation results, a simple kinetic model describing the transitions between the characteristic states was developed, and the rate constants for the essential transitions were estimated. During the folding process the system dwells in the bifurcation basin for a relatively short time before it proceeds to the native or latent state. We suggest that such a bifurcation may occur generally for proteins in which native and latent states have a sizable part of their structures in common. Moreover, there is the possibility of introducing changes in the system (e.g., mutations), which guide the system toward the native or misfolded state.     

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.     

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.     

Tertiary interactions determine the accuracy of RNA folding

RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild-type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30-200 s via nonnative intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild-type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.     

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.     

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.     

Role of cofactors in folding of the blue-copper protein azurin

Many proteins in living cells coordinate cofactors, such as metal ions, to attain their activity. Since the cofactors in such cases often can interact with their corresponding unfolded polypeptides in vitro, it is important to unravel how cofactors modulate protein folding. In this review, I will discuss the role of cofactors in folding of the blue-copper protein Pseudomonas aeruginosa azurin. In the case of both copper (Cu(II) and Cu(I)) and zinc (Zn(II)), the metal can bind to unfolded azurin. The residues involved in copper (Cu(II) and Cu(I)) coordination in the unfolded state have been identified as Cys112, His117, and Met121. The affinities of Cu(II), Cu(I), and Zn(II) are all higher for the folded than for the unfolded azurin polypeptide, resulting in metal stabilization of the native state as compared to the stability of apo-azurin. Cu(II), Zn(II), and several apo forms of azurin all fold in two-state kinetic reactions with roughly identical polypeptide-folding speeds. This suggests that the native-state beta-barrel topology, not cofactor interactions or thermodynamic stability, determines azurin's folding barrier. Nonetheless, copper binds much more rapidly (i.e., 4 orders of magnitude) to unfolded azurin than to folded azurin. Therefore, the fastest route to functional azurin is through copper binding before polypeptide folding; this sequence of events may be the relevant biological pathway.     

Kinetics of cooperative protein folding involving two separate conformational families

The master equation that describes the kinetics of protein folding is solved numerically for a portion of Staphylococcal Protein A by a Laplace transformation. The calculations are carried out with 50 local-minimum conformations belonging to two conformational families. The master equation allows for transitions among all the 50 conformations in the evolution toward the final folded equilibrium distribution of conformations. It is concluded that the native protein folds in a fast cooperative process. The global energy minimum of a native protein can be reached after a sufficiently long folding time regardless of the initial state and the existence of a large number of local energy minima. Conformations representing non-native states of the protein can transform to the native state even if they do not belong to the native conformational family. Given a starting conformation, the protein molecule can fold to its final conformation through different paths. Finally, when the folding reaches the equilibrium distribution, the protein molecule adopts a set of conformations in which the global minimum has the largest average probability.     

Investigation of folding in protamine by FTIR

The secondary structure of purified protamine, a non-specific DNA binding protein, was studied in solution at pH 4, 7 and 8 by FTIR. This permitted analysis of the folded form of the protein (acidic pH) as well as the folded conformers (neutral and basic pH). Hg(2+) was utilized to probe the accessibility of the free thiol groups (cysteine residues). The SH groups form when disulfides, which play the major role in stabilizing the conformation of this protein, are broken. It was possible to observe different conformational states in protamine as a function of pH and the presence of Hg(2+) via spectral changes primarily in the amide region. The results lead to the conclusion that protamine is not completely folded under conditions similar to those found in vivo (37 degrees , neutral pH, phosphate buffer and high protein concentration).     

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).     

Ultrafast folding of a computationally designed Trp-cage mutant: Trp2-cage

Miniproteins provide useful model systems for understanding the principles of protein folding and design. These proteins also serve as useful test cases for theories of protein folding, and their small size and ultrafast folding kinetics put them in a regime of size and time scales that is now becoming accessible to molecular dynamics simulations. Previous estimates have suggested the "speed limit" for folding is on the order of 1 mus. Here a computationally designed mutant of the 20-residue Trp-cage miniprotein, Trp2-cage, is presented. The Trp2-cage has greater stability than the parent and folds on the ultrafast time scale of 1 mICROs at room temperature, as determined from infrared temperature-jump experiments.