Folding Energy Landscape Research Articles

Multi-Scale Simulations of Proteins in Different Solvent Conditions

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulation in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics (AA-REMD) and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.

Size and Shape of Crowders Affect the Folding Landscape of Cytochrome C

We use computer simulation to investigate the effects of synthetic crowders on cytochrome c, a small single-domain protein with a cofactor heme. The folding energy landscape of cytochrome was computed in the presence of crowders with various sizes and shapes by using a coarse-grained protein model and the structure-based (Go-like) interactions. Our results demonstrated that given the same volume fraction of crowders, the stability of a folded protein inversely increases with the radius of crowders. In addition, a crowder with an anisotropic geometry imposes a greater stabilizing effect on the folded protein than isotropic crowders. This is in agreement with the predictions by the scaled particle theory. In addition, the distribution of contact formation between heme and cytochrome c protein was found to be varied by different types of crowders, demonstrating that the geometry of crowders may be one of the key factors for tuning heme-protein contact formation under cell-like conditions. Prospects of mixed crowders will be presented.

Unzip Single Protein Zippers using Optical Tweezers

Basic leucine zippers (bZIPs) are dimeric alpha-helical domains found in many transcription factors. They comprise coiled coil regions (zippers) that mediate specific protein dimerization, and basic regions that undergo disorder-to-order transitions to grip their cognate DNA binding sites. It remains unclear how folding of the two regions and binding of DNA are coupled. We investigated the detailed folding kinetics of a model bZIP domain derived from GCN4 transcriptional factor, using high-resolution optical tweezers. When pulled from the same amino- or carboxyl-terminals, the coiled coil alone folds and unfolds rapidly in a two-state manner, with little energy barrier. The folding kinetics of the full GCN4 bZIP domain is similar to the coiled coil alone. However, the presence of its binding site (AP1) induces a third new state corresponding to the bZIP-DNA complex. The probability of this new state critically depends upon the AP1 concentration, which reaches a maximum at an intermediate AP1 concentration. Results from our single-molecule manipulation experiments reveal a complex folding energy landscape even for a small protein domain that can be further tuned by their binding ligands.

Folding Studies of Beta-Strand-Containing Repeat Proteins Through Naturally-Occurring and Consensus-Designed Sequences

Repeat proteins, devoid of sequence-distant contacts observed in globular proteins, are ideal candidates for the dissection of local stability, nearest-neighbor contact parameters, cooperativity, and determination of folding energy landscapes. Recent studies on HEAT, TPR, Ankyrin, and Armadillo have contributed to the understanding of the folding of helical repeat proteins. Moreover, constructs composed of simplified “consensus” sequence representations of helical repeat proteins have been found to be well-behaved and highly stable, and facilitate energy dissection using simple nearest-neighbor models. In contrast, little progress has been made towards understanding the folding of β-sheet containing repeat. To determine the folding stability and cooperativity of these proteins, and to better understand the sequence determinants of structure and stability within these ubiquitous families, we have initiated studies on a series of LRR proteins of both naturally occurring and consensus-designed sequences. We find the LRR proteins PP32 and LC1 to be well-behaved, and to fold in a highly cooperative transition that is consistent with a two-state mechanism. NMR H/2H exchange shows the repeating β-strands on the concave surface of both proteins to be more protected than the rest of the molecules, and can be regarded as an exchange-resistant core, whereas the terminal caps and convex structural elements are more labile. However, truncations and sequence substitution demonstrate that the caps significantly influence stability and kinetics. To further simplify our analysis of β-sheet containing repeat protein folding, we designed consensus LRR sequences. On their own, these constructs are unfolded and/or aggregated. By fusing these consensus sequences with naturally occurring LRR protein YopM, we have obtained solublized, folded arrays that exhibit increased stability and drastically decreased unfolding and refolding rates with repeat number. Further studies are needed to dissect the complex folding pathways taken by these constructs.

Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The ΔΔG° values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol(-1). In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.

Protein Folding in the Cytoplasm and the Heat Shock Response

Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.

Overlap between folding and functional energy landscapes for adenylate kinase conformational change

Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK(e)) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK(e) has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AMP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK(e).

Nanosecond-Regime Correlation Time Scales for Equilibrium Protein Structural Fluctuations of Metal-Free Cytochrome c from Picosecond Time-Resolved Fluorescence Spectroscopy and the Dynamic Stokes Shift

We used picosecond time-resolved fluorescence spectroscopy to characterize the fluorescence Stokes shift (FSS) response function of metal-free (or free-base, fbCytc) cytochrome c under the solution conditions that favor the native states of ferricytochrome c (FeCytc) and Zn(II)-substituted cytochrome c (ZnCytc). The intrinsic porphyrin chromophore serves in these experiments as a fluorescent probe of the structural fluctuations of the surrounding protein and solvent. Demetalation of the porphyrin destabilizes the folded structure of cytochrome c owing to the loss of the axial metal-histidine and metal-methionine bonds. Thus, these experiments examine how the time scales detected in a dynamic solvation experiment in a chromoprotein report changes in the character of motion. The FSS response function in fbCytc in water and pH 7 is well described by a biexponential response over the 100 ps to 50 ns regime with time constants of 1.4 and 9.1 ns; under similar conditions, ZnCytc exhibits a biexponential FSS response with time constants of 250 ps and 1.5 ns [Lampa-Pastirk and Beck, J. Phys. Chem. B 2004, 108, 16288]. These time constants correspond, respectively, to the correlation time scales for motions of the hydrophobic core and the solvent-contact layer of the protein. Both of the time constants observed in fbCytc are further lengthened upon addition of glycerol to the external solvent so that a significant fraction of the protein dynamics is rendered effectively static on the fluorescence time scale. The solvation reorganization energy, the time-integrated Stokes shift of the fluorescence spectrum, is reduced by about a third to 33 cm(-1) in 50% glycerol from 43 cm(-1) in water. These results are interpreted structurally using a model for Brownian diffusive motion with thermally activated barrier crossings on the protein-folding energy landscape. The results suggest that the mean-squared deviations of the structural fluctuations exhibited by fbCytc are nearly a factor of 10 larger than those of ZnCytc. This conclusion is consistent with the suggestion that fbCytc assumes a dynamic, partially unfolded structure with some of the characteristics of a molten globule.

Structure and folding of a designed knotted protein

A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.

Local control of a disorder–order transition in 4E-BP1 underpins regulation of translation via eIF4E

The molecular mechanism underpinning regulation of eukaryotic translation initiation factor eIF4E by 4E-BP1 has remained unclear. We use isothermal calorimetry, circular dichroism, NMR, and computational modeling to analyze how the structure of the eIF4E-binding domain of 4E-BP1 determines its affinity for the dorsal face of eIF4E and thus the ability of this regulator to act as a competitive inhibitor. This work identifies the key role of solvent-facing amino acids in 4E-BP1 that are not directly engaged in interactions with eIF4E. These amino acid residues influence the propensity of the natively unfolded binding motif to fold into a conformation, including a stretch of α-helix, that is required for tight binding to eIF4E. In so doing, they contribute to a free energy landscape for 4E-BP1 folding that is poised so that phosphorylation of S65 at the C-terminal end of the helical region can modulate the propensity of folding, and thus regulate the overall free energy of 4E-BP1 binding to eIF4E, over a physiologically significant range. Thus, phosphorylation acts as an intramolecular structural modulator that biases the free energy landscape for the disorder-order transition of 4E-BP1 by destabilizing the α-helix to favor the unfolded form that cannot bind eIF4E. This type of order-disorder regulatory mechanism is likely to be relevant to other intermolecular regulatory phenomena in the cell.

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pKas allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pKa for Asp8 in the denatured state of wild-type, which is due to a nonnative interaction between Asp8 and Lys12. Interestingly, the simulation also shows a nonnative interaction between Asp8 and Glu48 in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.

The Plastic Energy Landscape of Protein Folding: A TRIANGULAR FOLDING MECHANISM WITH AN EQUILIBRIUM INTERMEDIATE FOR A SMALL PROTEIN DOMAIN

Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un)folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on- or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.

Two Amyloid States of the Prion Protein Display Significantly Different Folding Patterns

It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein. Several biophysical techniques including X-ray diffraction, CD, Fourier transform infrared spectroscopy (FTIR), hydrogen–deuterium exchange, proteinase K digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary, and quaternary structures. While both states displayed a 4. 8-Å meridional X-ray diffraction typical for amyloid cross-β-spines, they showed markedly different equatorial profiles, suggesting different folding pattern of β-strands. The experiments on hydrogen–deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-β-structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 °C, only local unfolding was revealed, while individual state-specific cross-β features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-β-structure. Both S- and R-fibrils, however, shared high conformational stability, arguing that the energy landscape for protein folding and aggregation can contain several deep free-energy minima.

BarMap: RNA folding on dynamic energy landscapes

Dynamical changes of RNA secondary structures play an important role in the function of many regulatory RNAs. Such kinetic effects, especially in time-variable and externally triggered systems, are usually investigated by means of extensive and expensive simulations of large sets of individual folding trajectories. Here we describe the theoretical foundations of a generic approach that not only allows the direct computation of approximate population densities but also reduces the efforts required to analyze the folding energy landscapes to a one-time preprocessing step. The basic idea is to consider the kinetics on individual landscapes and to model external triggers and environmental changes as small but discrete changes in the landscapes. A "barmap" links macrostates of temporally adjacent landscapes and defines the transfer of population densities from one "snapshot" to the next. Implemented in the BarMap software, this approach makes it feasible to study folding processes at the level of basins, saddle points, and barriers for many nonstationary scenarios, including temperature changes, cotranscriptional folding, refolding in consequence to degradation, and mechanically constrained kinetics, as in the case of the translocation of a polymer through a pore.

Multiscale investigation of chemical interference in proteins

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulations in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. An excessive amount of water molecules in all-atomistic molecular dynamics simulations often negatively impacts the sampling efficiency of some advanced sampling techniques such as the replica exchange method and it makes the investigation of chemical interferences on protein dynamics difficult. Thus, there is a need to develop an effective strategy that focuses on sampling structural changes in protein conformations rather than solvent molecule fluctuations. In this work, we address this issue by devising a multiscale simulation scheme (MultiSCAAL) that bridges the gap between all-atomistic molecular dynamics simulation and coarse-grained molecular simulation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side-chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.

Infrared Signature and Folding Dynamics of a Helical β-Peptide

Synthetic foldamers consisting of beta-amino acids offer excellent model systems for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a beta-peptide that folds into a 14-helical structure in water. We find that the T-jump induced relaxation kinetics of this peptide occur on the nanosecond time scale and are noticeably slower than those of alanine-based alpha-helical peptides, and additionally, the relaxation rates show a weaker dependence on temperature. These differences appear to indicate that the folding energy landscapes of these peptides are different. In addition, we find that the amide I' band of this beta-peptide exhibits a sharp feature at approximately 1612 cm(-1), which we believe is a distinct infrared reporter of 14-helix.

The Folding Energy Landscape and Free Energy Excitations of Cytochrome c

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Because of the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case, following principles different from those used to describe the folding of proteins without cofactors. Recent investigations, however, demonstrate that common models describing folding for many proteins work well for cytochrome c when heme is explicitly introduced, generally providing results that agree with experimental observations. In this Account, we first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Because attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as "perfectly funneled". In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are denoted sources of "frustration", which cause specific transient intermediates to be populated. Native structure-based models do, however, include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold so that folding can proceed. The ability of native structure-based models to capture this kind of motion is partly responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe the sequence of folding events for cytochrome c well (as inferred from hydrogen-exchange experiments), thereby justifying their use as a starting point. At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. Here, alternate folding pathways are a result of "chemical frustration". This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. Beginning with native structure-based terms, we construct more complex models by adding chemical frustration. These more complex models only modestly perturb the energy landscape, which remains, overall, well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. The success of models based on funneled energy landscapes suggest that cytochrome c folding is driven primarily by native contacts. The presence of heme appears to add chemical complexity to the folding process, but it does not require fundamental modification of the general principles used to describe folding. Moreover, its added complexity provides a valuable means of probing the folding energy landscape in greater detail than is possible with simpler systems.

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin