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.     

On the origin of NMR dipolar waves in transient helical elements of partially folded proteins

The presence of dipolar coupling waves within helical elements of proteins implies an effective tilt of the main axis of the helical element relative to the magnetic field. Here, we investigate the origin of dipolar waves observed in helical elements of partially folded proteins. We find that the dipolar waves result from an effective tilt of the helix relative to the alignment axis that is determined by the directionality of the unfolded chains projected from the helix caps. The amplitude and phase of the dipolar wave depend in a predictable way on helix length, providing direct insight into helix stability, nucleation, and fraying in partially folded proteins.     

Molecular simulation of protein dynamics in nanopores. I. Stability and folding

Discontinuous molecular dynamics simulations, together with the protein intermediate resolution model, an intermediate-resolution model of proteins, are used to carry out several microsecond-long simulations and study folding transition and stability of alpha-de novo-designed proteins in slit nanopores. Both attractive and repulsive interaction potentials between the proteins and the pore walls are considered. Near the folding temperature T(f) and in the presence of the attractive potential, the proteins undergo a repeating sequence of folding/partially folding/unfolding transitions, with T(f) decreasing with decreasing pore sizes. The unfolded states may even be completely adsorbed on the pore's walls with a negative potential energy. In such pores the energetic effects dominate the entropic effects. As a result, the unfolded state is stabilized, with a folding temperature T(f) which is lower than its value in the bulk and that, compared with the bulk, the folding rate decreases. The opposite is true in the presence of a repulsive interaction potential between the proteins and the walls. Moreover, for short proteins in very tight pores with attractive walls, there exists an unfolded state with only one alpha-helical hydrogen bond and an energy nearly equal to that of the folded state. The proteins have, however, high entropies, implying that they cannot fold onto their native structure, whereas in the presence of repulsive walls the proteins do attain their native structure. There is a pronounced asymmetry between the two termini of the protein with respect to their interaction with the pore walls. The effect of a variety of factors, including the pore size and the proteins' length, as well as the temperature, is studied in detail.     

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.     

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.     

A molecular dynamics study of the correlations between solvent-accessible surface, molecular volume, and folding state

We analyzed the correlations between molecular volume, solvent-accessible surface, and folding state (secondary structure content) for unfolded conformers of alpha (holo- and apomyoglobin) and beta (retinal-binding protein) proteins and a small water-soluble alanine-rich alpha-helical peptide. Conformers with different degrees of folding were obtained using molecular dynamics at constant temperature and pressure with implicit solvent (dielectric constant adjustment) for all four systems and with explicit solvent for the single helix peptide. Our results support the view that unfolded conformations are not necessary extended, that volume variation is not a good indication of folding state and that the simple model of water penetrating the interior of the protein does not explain the increase in volume upon unfolding.     

Protein folding in a reverse micelle environment: the role of confinement and dehydration

Characterization of the molecular interactions that stabilize the folded state of proteins including hydrogen bond formation, solvation, molecular crowding, and interaction with membrane environments is a fundamental goal of theoretical biophysics. Inspired by recent experimental studies by Gai and co-workers, we have used molecular dynamics simulations to explore the structure and dynamics of the alanine-rich AKA(2) peptide in bulk solution and in a reverse micelle environment. The simulated structure of the reverse micelle shows substantial deviations from a spherical geometry. The AKA(2) peptide is observed to (1) remain in a helical conformation within a spherically constrained reverse micelle and (2) partially unfold when simulated in an unconstrained reverse micelle environment, in agreement with experiment. While aqueous solvation is found to stabilize the N- and C-termini random coil portions of the peptide, the helical core region is stabilized by significant interaction between the nonpolar surface of the helix and the aliphatic chains of the AOT surfactant. The results suggest an important role for nonpolar peptide-surfactant and peptide-lipid interactions in stabilizing helical geometries of peptides in reverse micelle environments.     

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.     

Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

Anion Recognition by Aliphatic Helical Oligoureas

Anion binding properties of neutral helical foldamers consisting of urea type units in their backbone have been investigated. ¹H NMR titration studies in various organic solvents including DMSO suggest that the interaction between aliphatic oligoureas and anions (CH₃COO^?, H₂PO₄^?, Cl^?) is site‐specific, as it largely involves the urea NHs located at the terminal end of the helix (positive pole of the helix), which do not participate to the helical intramolecular hydrogen‐bonding network. This mode of binding parallels that found in proteins in which anion‐binding sites are frequently found at the N‐terminus of an α‐helix. ¹H NMR studies suggest that the helix of oligoureas remains largely folded upon anion binding, even in the presence of a large excess of the anion. This study points to potentially useful applications of oligourea helices for the selective recognition of small guest molecules.     

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.     

Molecular dynamics simulation of folding of a short helical peptide with many charged residues

A molecular dynamics simulation of the folding of conantokin-T (con-T), a short helical peptide with 5 helical turns of 21 amino acids with 10 charged residues, was carried out to examine folding pathways for this peptide and to predict the folding rate. In the 18 trajectories run at 300 K, 16 trajectories folded, with an averaged folding time of approximately 50 ns. Two trajectories did not fold in up to 200 ns simulation. The folded structure in folded trajectories is in good agreement with experimental structure. An analysis of the trajectories showed that, at the beginning of a few nanoseconds, helix formation started from residues 5-9 with assistance of a hydrophobic clustering involving Tyr5, Met8, and Leu9. The peptide formed a U-shape mainly due to charge-charge interactions between charged residues at the N- and C-terminus segments. In the next approximately 10 ns, several nonnative charge-charge interactions were broken and nonnative Gla10-Lys18 (this denotes a salt bridge between Gal10 and Lys18) and/or Gla10-Lys19 interactions appeared more frequently in this folding step and the peptide became a fishhook J-shape. From this structure, the peptide folded to the folded state in 7 of all 16 folded trajectories in approximately 15 ns. Alternatively, in approximately 30 ns, the con-T went to a conformation in an L-shape with 4 helical turns and a kink at the Arg13 and Gla14 segment in the other 9 trajectories. Con-T in the L-shape then required another approximately 15 ns to fold into the folded state. In addition, in overall folding times, the former 7 trajectories folded faster with the total folding times all shorter than 45 ns, while the latter 9 trajectories folded at a time longer than 45 ns, resulting in an average folding time of approximately 50 ns. Two major folding intermediates found in 2 nonfolded trajectories are stabilized by charge clusters of 5 and 6 charged residues, respectively. With inclusion of friction and solvent-solvent interactions, which were ignored in the present GB/SA solvation model, the folding time obtained above should be multiplied by a factor of 1.25-1.7 according to a previous, similar simulation study. This results in a folding time of 65-105 ns, slightly shorter than the folding time of 127 ns for an alanine-based peptide of the same length. This suggests that the energy barrier of folding for this type of peptides with many charged residues is slightly lower than alanine-based helical peptides by less than 1 kcal/mol.     

Is there a unique melting temperature for two-state proteins?

Thermal unfolding (or folding) in many proteins occurs in an apparent two-state manner, suggesting that only two states, unfolded and folded, are populated. At the melting temperature, Tm, the two states coexist. Using lattice models with side chains we show that individual residues become structured at temperatures that deviate from Tm, which implies that partially folded conformations make substantial contribution to thermodynamic properties of two-state proteins. We also find that the folding cooperativity for a given residue is linked to its accessible surface area. These results are consistent with the experiments on GCN4-like zipper peptide, which showed that local melting temperatures differ from Tm. Analysis of thermal unfolding of six proteins shows that deltaT/Tm approximately N(-1), where deltaT is the transition width and N is the number of residues. This scaling allows us to conclude that, when corrected for finite size effects, folding cooperativity can be captured using coarse grained models.     

Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.     

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.     

Helical Folding Competing with Unfolded Aggregation in Phenylene Ethynylene Foldamers

The folding and aggregation behavior of a pair of oligo(phenylene ethynylene) (OPE) foldamers are investigated by means of UV/Vis absorption and circular dichroism spectroscopy. With identical OPE backbones, two foldamers, 1 with alkyl side groups and 2 with triethylene glycol side chains, manifest similar helical conformations in solutions in n‐hexane and methanol, respectively. However, disparate and competing folding and aggregation processes are observed in alternative solvents. In cyclohexane, oligomer 1 initially adopts the helical conformation, but the self‐aggregation of unfolded chains, as a minor component, gradually drives the folding–unfolding transition eventually to the unfolded aggregate state completely. In contrast, in aqueous solution (CH₃OH/H₂O) both folded and unfolded oligomer 2 appear to undergo self‐association; aggregates of the folded chains are thermodynamically more stable. In solutions with a high H₂O content, self‐aggregation among unfolded oligomers is kinetically favored; these oligomers very slowly transform into aggregates of helical structures with greater thermodynamic stability. The folded–unfolded conformational switch thus takes place with the free (nonaggregated) molecules, and the very slow folding transition is due to the low concentration of molecularly dispersed oligomers.     

Molecular weight recognition in the multiple-stranded helix of a synthetic polymer without specific monomer-monomer interaction

Stereoregular isotactic and syndiotactic poly(methyl methacrylate)s (it- and st-PMMAs) are known to form a multiple-stranded complementary helix, so-called stereocomplex (SC) through van der Waals interactions, which is a rare example of helical supramolecular structures formed by a commodity polymer. In this study, we prepared SCs by using uniform it- and st-PMMAs and those with a narrow molecular weight distribution having different molecular weights and investigated their structures in detail using high-resolution atomic force microscopy as a function of the molecular weight and molecular weight distribution of the component PMMAs. We found that complementary it- and st-PMMAs with the longer molecular length determine the total length of the SC, and molecules of the shorter component associate until they fill up or cover the longer component. These observations support a supramolecular triple-stranded helical structure of the SCs composed of a double-stranded helix of two intertwined it-PMMA chains included in a single helix of st-PMMA, and this triple-stranded helix model of the SCs appears to be applicable to the it- and st-PMMAs having a wide range of molecular weights we employed in this study. In homogeneous double-stranded helices of it-PMMA, it has been found that, in mixtures of two it-PMMAs with different molecular weights, chains of the same molecular weight selectively form a double-stranded it-PMMA helix, or recognize the molecular weights of each other ("molecular sorting"). We thus demonstrate that molecular weight recognition is possible, without any specific interaction between monomer units, through the formation of a topological multiple-stranded helical structure based upon van der Waals interaction.     

Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.     

An Off‐Pathway Folding Intermediate of an Acyl Carrier Protein Domain Coexists with the Folded and Unfolded States under Native Conditions

A protein can exist in multiple states under native conditions and those states with low populations are often critical to biological function and self‐assembly. To investigate the role of the minor states of an acyl carrier protein, NMR techniques were applied to determine the number of minor states and characterize their structures and kinetics. The acyl carrier protein from Micromonospora echinospora was found to exist in one major folded state (95.2 %), one unfolded state (4.1 %), and one intermediate state (0.7 %) under native conditions. The three states are in dynamic equilibrium and the intermediate state very likely adopts a native‐like structure and is an off‐pathway folding product. The intermediate state may mediate the formation of oligomers in vitro and play an important role in the recognition of partner enzymes in vivo.     

Folding dynamics of Trp-cage in the presence of chemical interference and macromolecular crowding. I

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.