Confinement in nanopores can destabilize α-helix folding proteins and stabilize the β structures

Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an α-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller α-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the β structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the α → β conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight confinement. For in silico studies of protein folding in confined media, use of non-Go potentials may be more appropriate.     

Polar confinement modulates solvation behavior of methane molecules

Polar confinement induces an amorphous solidlike state of water characterized by an orientational correlation time longer than hundreds of picoseconds and significant structural disorder. Solvation behavior of methane molecules is dramatically modulated under polar confinement. Moreover our simulations indicate that the charges equivalent to those borne by atoms of amino acids could generate an electric field which is strong enough to stimulate the phase transition of water. In our results, polar confinement is found to be more capable of aggregating hydrophobic molecules. This study raises an interesting mechanism by which the cagelike structure of the Escherichia coli chaperonin GroEL and the cochaperonin GroES complex helps protein folding.     

A role for confined water in chaperonin function

Chaperonins engulf other proteins and accelerate their folding by an unknown mechanism. Here, we combine all-atom molecular dynamics simulations with data from experimental assays of the activity of the bacterial chaperonin GroEL to demonstrate that a chaperonin's ability to facilitate folding is correlated with the affinity of its interior surface for water. Our results suggest a novel view of the behavior of confined water for models of in vivo protein folding scenarios.     

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.     

Helical Folding of Conjugated Oligo(phenyleneethynylene): Chain‐Length Dependence,Solvent Effects,and Intermolecular Assembly

As a representative folding system that features a conjugated backbone, a series of monodispersed (o‐phenyleneethynylene)‐alt‐(p‐phenyleneethynylene) (PE) oligomers of varied chain length and different side chains were studied. Molecules with the same backbone but different side‐chain structures were shown to exhibit similar helical conformations in respectively suitable solvents. Specifically, oligomers with dodecyloxy side chains folded into the helical structure in apolar aliphatic solvents, whereas an analogous oligomer with tri(ethylene glycol) (Tg) side chains adopted the same conformation in polar solvents. The fact that the oligomers with the same backbone manifested a similar folded conformation independent of side chains and the nature of the solvent confirmed the concept that the driving force for folding was the intramolecular aromatic stacking and solvophobic interactions. Although all were capable of inducing folding, different solvents were shown to bestow slightly varied folding stability. The chain‐length dependence study revealed a nonlinear correlation between the folding stability with backbone chain length. A critical size of approximately 10 PE units was identified for the system, beyond which folding occurred. This observation corroborated the helical nature of the folded structure. Remarkably, based on the absorption and emission spectra, the effective conjugation length of the system extended more effectively under the folded state than under random conformations. Moreover, as evidenced by the optical spectra and dynamic light‐scattering studies, intermolecular association took place among the helical oligomers with Tg side chains in aqueous solution. The demonstrated ability of such a conjugated foldamer in self‐assembling into hierarchical supramolecular structures promises application potential for the system.     

Use of selective Trp side chain labeling to characterize protein-protein and protein-ligand interactions by NMR spectroscopy

Recent studies on amino acid occurrence in protein binding sites suggest that only a reduced number of residues are responsible for most interaction energy in protein-protein and protein-ligand interactions. Above all, tryptophan (Trp) seems to be the most frequent residue in protein's hot spots. Here we report a novel, efficient, and cost-effective method to selectively incorporate specific isotope labels into the side chains of Trp residues in recombinant proteins. We show that the method proposed allows selective NMR observation of Trp side chains that enables studies of ligand binding, protein-protein interactions, hydrogen binding, protein folding, and side chain dynamics. Examples with the protein BIR3 will be given.     

Simulation of chaperonin effect on protein folding: a shift from nucleation-condensation to framework mechanism

The iterative annealing mechanism (IAM) of chaperonin-assisted protein folding is explored in a framework of a well-established coarse-grained protein modeling tool, which enables the study of protein dynamics in a time-scale well beyond classical all-atom molecular mechanics. The chaperonin mechanism of action is simulated for two paradigm systems of protein folding, B domain of protein A (BdpA) and B1 domain of protein G (GB1), and compared to chaperonin-free simulations presented here for BdpA and recently published for GB1. The prediction of the BdpA transition state ensemble (TSE) is in perfect agreement with experimental findings. It is shown that periodic distortion of the polypeptide chains by hydrophobic chaperonin interactions can promote rapid folding and leads to a decrease in folding temperature. It is also demonstrated how chaperonin action prevents kinetically trapped conformations and modulates the observed folding mechanisms from nucleation-condensation to a more framework-like.     

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.     

Design and application of basic amino acids displaying enhanced hydrophobicity

Three noncoding basic amino acids, mono-, di-, and trimethyldiaminopropionic acid (mmdap, dmdap, and tmdap), have been synthesized for use in protein design. Covalent modification of a diaminopropionic acid (dap) side chain with an increasing number of methyl moieties results in a family of residues displaying short basic side chains with varying degrees of enhanced hydrophobic character. These residues may be used to introduce charged/polar interactions into the confining hydrophobic interior or interfacial spaces of proteins. As a demonstration of their utility, the ability of these residues to promote interior salt bridge formation at the helix/helix interface of GCN4-p1, a dimeric two-stranded coiled coil, was assessed. Heterodimerization mediated by buried salt bridge formation between a GCN4-based peptide containing either mmdap, dmdap, or tmdap at position 16 and an analogous peptide containing aspartic acid at the same position was studied. Mmdap-derived heterodimers are 0.5 kcal/mol more stable than the corresponding dap-derived heterodimers. This result indicates that the addition of one methyl group to the dap side chain can stabilize the heterodimeric fold. The stabilization can most likely be attributed to a decrease in the desolvation penalty incurred upon folding as well as enhanced van der Waals contacts in the folded state. The addition of three methyl groups to the dap side chain results in heterodimers that are significantly less stable than the corresponding dap-derived heterodimers, suggesting that increased steric bulk is not well accommodated in the interior of this protein. Unexpectedly, the addition of two methyl groups leads to homotrimerization of the dmdap-peptide. The resulting trimer is relatively stable (DeltaG(37)( degrees )(C) degrees = 11.8 kcal/mol) and undergoes cooperative thermal unfolding. The GCN4-p1 system exemplifies how small incremental changes in size and hydrophobicity can alter the folding preferences of a protein. Generally, this versatile suite of residues can be utilized in any protein and offer new options to the protein chemist.     

The burial of solvent-accessible surface area is a predictor of polypeptide folding and misfolding as a function of chain elongation

The hydrophobic effect is a major driving force in all chemical and biological events involving chain collapse in aqueous solution. Here, we show that the burial of nonpolar solvent-accessible surface area (NSASA) is a powerful criterion to predict the folding and misfolding behavior of small single-domain proteins as a function of chain elongation. This bears fundamental implications for co- and post-translational protein folding in the cell and for understanding the interplay between noncovalent interactions and formation of native-like structure and topology. Comparison between the fraction of NSASA in fully unfolded and folded elongating chains shows that efficient burial of nonpolar surface area is preferentially achieved only when the polypeptide chain is almost complete. This effect has no preferential vectorial character in that it is present upon elongation from both the N and C termini. For incomplete chains that do not have the ability to fold and bury nonpolar surface intramolecularly, the overall hydrophobic nature of the polypeptide chain (expressed as FBA, i.e., fractional buried surface area per residue) dictates the tendency toward misfolding and self-association. N-terminal chains characterized by FBA exceeding 0.73 are likely to misfold and aggregate, if unable to fold intramolecularly.     

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.     

Modifying the OPLS-AA force field to improve hydration free energies for several amino acid side chains using new atomic charges and an off-plane charge model for aromatic residues

The hydration free energies of amino acid side chains are an important determinant of processes that involve partitioning between different environments, including protein folding, protein complex formation, and protein-membrane interactions. Several recent papers have shown that calculated hydration free energies for polar and aromatic residues (Trp, His, Tyr, Asn, Gln, Asp, Glu) in several common molecular dynamics force fields differ significantly from experimentally measured values. We have attempted to improve the hydration energies for these residues by modifying the partial charges of the OPLS-AA force field based on natural population analysis of density functional theory calculations. The resulting differences between calculated hydration free energies and experimental results for the seven side chain analogs are less than 0.1 kcal/mol. Simulations of the synthetic Trp-rich peptide Trpzip2 show that the new charges lead to significantly improved geometries for interacting Trp-side chains. We also investigated an off-plane charge model for aromatic rings that more closely mimics their electronic configuration. This model results in an improved free energy of hydration for Trp and a somewhat altered benzene-sodium potential of mean force with a more favorable energy for direct benzene-sodium contact.     

How ion properties determine the stability of a lipase enzyme in ionic liquids: a molecular dynamics study

The influence of eight different ionic liquid (IL) solvents on the stability of the lipase Candida antarctica lipase B (CAL-B) is investigated with molecular dynamics (MD) simulations. Considered ILs contain cations that are based either on imidazolium or guanidinium as well as nitrate, tetrafluoroborate or hexafluorophosphate anions. Partial unfolding of CAL-B is observed during high-temperature MD simulations and related changes of CAL-B regarding its radius of gyration, surface area, secondary structure, amount of solvent close to the backbone and interaction strength with the ILs are evaluated. CAL-B stability is influenced primarily by anions in the order NO(3)(-)? BF(4)(-) < PF(6)(-) of increasing stability, which agrees with experiments. Cations influence protein stability less than anions but still substantially. Long decyl side chains, polar methoxy groups and guanidinium-based cations destabilize CAL-B more than short methyl groups, other non-polar groups and imidazolium-based cations, respectively. Two distinct causes for CAL-B destabilization are identified: a destabilization of the protein surface is facilitated mostly by strong Coulomb interactions of CAL-B with anions that exhibit a localized charge and strong polarization as well as with polar cation groups. Surface instability is characterized by an unraveling of α-helices and an increase of surface area, radius of gyration and protein-IL total interaction strength of CAL-B, all of which describe a destabilization of the folded protein state. On the other hand, a destabilization of the protein core is facilitated when direct core-IL interactions are feasible. This is the case when long alkyl chains are involved or when particularly hydrophobic ILs induce major conformational changes that enable ILs direct access to the protein core. This core instability is characterized by a disintegration of β-sheets, diffusion of ions into CAL-B and increasing protein-IL van der Waals interactions. This process describes a stabilization of the unfolded protein state. Both of these processes reduce the folding free energy and thus destabilize CAL-B. The results of this work clarify the impact of ions on CAL-B stabilization. An extrapolation of the observed trends enables proposing novel ILs in which protein stability could be enhanced further.     

Putting a lid on protein folding: structure and function of the co-chaperonin,GroES

The co-chaperonin GroES is an essential partner in protein folding mediated by the chaperonin, GroEL. Two recent crystal structures of GroES provide a structural basis to understand how GroES forms the lid on the folding-active cis ternary complex, and how the GroEL-GroES complex enhances folding.     

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.     

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.     

Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy

Hydrophobic interactions play a major role in binding non-native substrate proteins in the central cavity of the bacterial chaperonin GroEL. The sequence of local conformational changes by which GroEL and its cofactor GroES assist protein folding can be explored using the polarity-sensitive fluorescence probe Nile Red. A specific single-cysteine mutant of GroEL (Cys261), whose cysteine is located inside the central cavity at the apical region of the protein, was covalently labeled with synthetically prepared Nile Red maleimide (NR). Bulk fluorescence spectra of Cys261-NR were measured to examine the effects of binding of the stringent substrate, malate dehydrogenase (MDH), GroES, and nucleotide on the local environment of the probe. After binding denatured substrate, the fluorescence intensity increased by 32 +/- 7%, suggesting enhanced hydrophobicity at the position of the label. On the other hand, in the presence of ATP, the fluorescence intensity decreased by 13 +/- 3%, implying increased local polarity. To explore the sequence of local polarity changes, substrate, GroES, and various nucleotides were added in different orders; the resulting changes in emission intensity provide insight into the sequence of conformational changes occurring during GroEL-mediated protein folding.     

Influence of variation of a side chain on the folding equilibrium of a β‐peptide: Limitations of one‐step perturbation

In a recent study (Lin et al., Helv. Chim. Acta 2011, 94 , 597), the one‐step perturbation method was applied to tackle a challenging computational problem, that is, the calculation of the folding free enthalpies ΔG_F,U of six hepta‐β‐peptides with different, Ala, Val, Leu, Ile, Ser, or Thr, side chains in the fifth residue. The ΔG_F,U values obtained using one‐step perturbation based on a single molecular dynamics simulation of a judiciously chosen reference state with soft‐core atoms in the side chain of the fifth residue showed an overall accuracy of about k_BT for the four peptides with nonpolar side chains, but twice as large deviations were observed for the peptides with polar side chains. Here, alternative reference‐state Hamiltonians that better cover the conformational space relevant to these peptides are investigated, and post simulation rotational sampling of the χ₁ and χ₂ torsional angles of the fifth residue is carried out to sample different orientations of the side chain. A reference state with rather soft atoms yields accurate ΔG_F,U values for the peptides with the Ser and Thr side chains, but it failed to correctly predict the folding free enthalpy for one peptide with a nonpolar side chain, that is, Leu. Based on the results and those of earlier studies, possible ways to improve the accuracy of the efficient one‐step perturbation technique to compute free enthalpies of folding are discussed. © 2013 Wiley Periodicals, Inc.     

Effects of polar group saturation on physical gelation of amphiphilic polymer solutions

Monte Carlo simulation on the basis of the comblike coarse grained nonpolar/polar (NP) model has been carried out to study the polar group saturation effect on physical gelation of amphiphilic polymer solutions. The effects of polar group saturation due to hydrogen bonding or ion bridging on the sol-gel phase diagram, microstructure of aggregates, and chain conformation of amphiphilic polymer solutions under four different solvent conditions to either the nonpolar backbone or the polar side chain in amphiphilic polymer chains have been investigated. It is found that an increase of polar group saturation results in a monotonically decreased critical concentration of gelation point, which can be qualitatively supported by the dynamic rheological measurements on pectin aqueous solutions. Furthermore, various solvent conditions to either the backbone or the side chain have significant impact on both chain conformation and microstructure of aggregates. When the solvent is repulsive to the nonpolar backbone but attractive to the polar side chain, the polymer chains are collapsed, and the gelation follows the mechanism of colloidal packing; at the other solvent conditions, the gelation follows the mechanism of random aggregation.     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.