Folding helical proteins in explicit solvent using dihedral-biased tempering

Using a single-trajectory-based tempering method with a high-temperature dihedral bias, we repeatedly folded four helical proteins [α₃D (PDB ID: 2A3D, 73 residues), α₃W (1LQ7, 67 residues), Fap1-NR_α (2KUB, 81 residues) and S-836 (2JUA, 102 residues)] and some of the mutants in explicit solvent within several microseconds. The lowest root-mean-square deviations of backbone atoms from the experimentally determined structures were 1.9, 1.4, 1.0, and 2.1 Å, respectively. Cluster analyses of folding trajectories showed the native conformation usually occupied the most populated cluster. The simulation protocol can be applied to large-scale simulations of other helical proteins on commonly accessible computing platforms.     

The dual-basin landscape in GFP folding

Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a “locking” mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above.     

Local conformational dynamics in α-helices measured by fast triplet transfer

Coupling fast triplet–triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix–coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in α-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 μs at 5 °C. Local helix formation occurs with a time constant of ≈400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with position-independent elementary time constants of ≈50 ns for the addition and of ≈65 ns for the removal of an α-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.     

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH⁰ = 35 ± 4 kJ/mol) which is nearly compensated in free energy by the entropy change (ΔS⁰ = 112 ± 20 J·mol^-1·K^-1). The unlocking reaction has a time constant of about 1 μs at 5 °C and is enthalpy-limited with an activation energy of 32 ± 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 × 10¹¹ s^-1. In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 ± 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.     

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous three-state proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of Φ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state.     

Energy landscape and multiroute folding of topologically complex proteins adenylate kinase and 2ouf-knot

While fast folding of small proteins has been relatively well characterized by experiments and theories, much less is known for slow folding of larger proteins, for which recent experiments suggested quite complex and rich folding behaviors. Here, we address how the energy landscape theory can be applied to these slow folding reactions. Combining the perfect-funnel approximation with a multiscale method, we first extended our previous atomic-interaction based coarse grained (AICG) model to take into account local flexibility of protein molecules. Using this model, we then investigated the energy landscapes and folding routes of two proteins with complex topologies: a multidomain protein adenylate kinase (AKE) and a knotted protein 2ouf-knot. In the AKE folding, consistent with experimental results, the kinetic free energy surface showed several substates between the fully unfolded and native states. We characterized the structural features of these substates and transitions among them, finding temperature-dependent multiroute folding. For protein 2ouf-knot, we found that the improved atomic-interaction based coarse-grained model can spontaneously tie a knot and fold the protein with a probability up to 96%. The computed folding rate of the knotted protein was much slower than that of its unknotted counterpart, in agreement with experimental findings. Similar to the AKE case, the 2ouf-knot folding exhibited several substates and transitions among them. Interestingly, we found a dead-end substate that lacks the knot, thus suggesting backtracking mechanisms.     

Stabilization of a protein conferred by an increase in folded state entropy

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.     

Twin-arginine translocase mutations that suppress folding quality control and permit export of misfolded substrate proteins

The bacterial twin-arginine translocation (Tat) pathway facilitates the transport of correctly folded proteins across the tightly sealed cytoplasmic membrane. Here, we report the isolation and characterization of suppressor mutations in the Tat translocase that allow export of misfolded proteins, which form structures that are not normally tolerated by the wild-type translocase. Selection of suppressors was enabled by a genetic assay that effectively linked in vivo folding and stability of a test protein with Tat export efficiency of a selectable marker protein, namely TEM-1 β-lactamase. By using a test protein named α(3)B-a designed three-helix-bundle protein that forms collapsed, stable molten globules but lacks a uniquely folded structure-translocase mutants that rescued export of this protein were readily identified. Each mutant translocase still efficiently exported folded substrate proteins, indicating that the substrate specificity of suppressors was relaxed but not strictly altered. A subset of the suppressors could also export other misfolded proteins, such as the aggregation-prone α(3)A protein and reduced alkaline phosphatase. Importantly, the isolation of genetic suppressors that inactivate the Tat quality-control mechanism provides direct evidence for the participation of the Tat translocase in structural proofreading of substrate proteins and reveals epitopes in the translocase that are important for this process.     

Hsp70 biases the folding pathways of client proteins

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a ¹H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.The 70-kDa heat shock protein (Hsp70) chaperone system is an important component of the cellular proteostasis machinery, serving as a central hub to channel client proteins along folding, refolding, maturation, disaggregation, and proteolytic pathways in cooperation with other chaperone assemblies such as Hsp90, Hsp104, and GroEL/ES (1–3). Central to Hsp70 function is its ATP-dependent interaction with client proteins, facilitated by Hsp40 cochaperones and nucleotide exchange factors (NEFs) (4). Hsp70 is a weak ATPase that recognizes and binds substrates at sites containing large aliphatic hydrophobic residues, Ile, Leu, and Val, flanked by positively charged amino acids such as Arg and Lys (5). Initial binding of substrate to the ATP-form of Hsp70 can occur directly, or via Hsp40, with rapid on/off kinetics that give rise to a weak overall affinity for the interaction. Subsequent ATP hydrolysis, stimulated by interactions with Hsp40 and substrate, leads to a large conformational change in the chaperone, locking the substrate in the Hsp70 bound state. The resulting complex is of high affinity with slow substrate on/off rates (2).Escherichia coli DnaK is the best studied of Hsp70 chaperones. It is a 70-kDa protein comprised of an N-terminal ATPase and a C-terminal substrate binding domain that communicate allosterically to couple ATP hydrolysis with substrate binding (3). High-resolution structures of ADP- (6) and ATP-DnaK (7, 8) establish that these two domains dock on to one another in the ATP-DnaK state, but become detached from each other in the ADP-bound form. In contrast to the detailed structural studies characterizing DnaK, little atomic resolution data are available on the conformation of folding-competent client proteins in the DnaK-bound state. It is known that DnaK binds substrates in a globally unfolded conformation with varying degrees of local residual native and nonnative secondary structure (9–13). However, whether stable or transient long-range interactions are present in DnaK-bound client proteins remains an open question that has relevance for understanding the function of DnaK in substrate refolding and disaggregation. For example, it has been shown that DnaK, in concert with cochaperones DnaJ (Hsp40) and GrpE (NEF), converts misfolded luciferase to a globally unfolded, yet folding-competent, conformation (14). Furthermore, the DnaK chaperone system is thought to “loosen” aggregated proteins for subsequent disaggregation by ClpB or proteolysis by ClpXP (15). Aggregated and misfolded proteins are stabilized by native and nonnative tertiary contacts and characterizing the extent to which these interactions either persist or are modified upon DnaK binding will provide insights into the mechanism by which DnaK carries out its myriad of important functions.Here we probe the existence of (transient) long-range tertiary interactions in a folding competent substrate, the human telomere repeat binding factor (hTRF1), which is globally unfolded when bound to DnaK (13), using paramagnetic relaxation enhancement (PRE) NMR spectroscopy. To evaluate whether DnaK binding modifies the energy landscape of the substrate in a way that affects long-range interactions in the unfolded state, we have recorded PREs in the unbound, unfolded conformation of hTRF1 under identical conditions to those used for studies of DnaK-bound hTRF1. The unfolded state of hTRF1 in water (referred to in what follows as uw-hTRF1) is only sparsely populated and transiently formed, rendering it invisible to standard NMR studies. However, the signal from the invisible state can be amplified using chemical exchange saturation transfer (CEST) and read out through the visible, folded hTRF1 state. We thus developed a ¹H-based CEST experiment that facilitates measurement of PREs in uw-hTRF1. A comparison of PREs in this state with those in the hTRF1-DnaK bound conformation establishes that the large PREs in uw-hTRF1 are significantly reduced on DnaK binding, and the extent of residual long-range interactions in the DnaK-bound form is similar to hTRF1 denatured in 4 M urea. Taken together, our results suggest that DnaK may be able to modify the folding pathways of protein substrates by significantly influencing their tertiary structural tendencies in the bound conformation.     

Induction of DNA damage by oxidised amino acids and proteins

Exposure of amino acids, peptides and proteins to radicals in the presence of O₂generates hydroperoxides in a dose-dependent manner. These hydroperoxides are stable in the absence of exogenous catalysts (e.g. heat, light, redox-active transition metal ions), but decompose rapidly in the presence of these agents to give a variety of radicals including alkoxyl (RO^•), peroxyl (ROO^•) and carbon-centred (R^•) species. These radicals are shown to react with DNA to give DNA-protein cross-links and single strand breaks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anomalous behavior of water inside the SecY translocon

The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359–E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a “primed” open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182–17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.The heterotrimeric SecY translocon complex (SecYEG in bacteria, SecYEβ in archaea, Sec61αβγ in eukaryotes) is required for the cotranslational assembly of membrane proteins and the secretion of soluble proteins (1–3). The SecY subunit (Fig. 1A) has 10 transmembrane helices comprised of two five-helix domains related by pseudo-twofold symmetry around an axis parallel to the membrane (4–8). These helices form an hourglass-shaped water-filled pore that spans the membrane. The so-called hydrophobic pore ring (HR) comprised of six hydrophobic residues forms the narrowest part of the hourglass, located near the bilayer center. Sitting just above the ring on the extracellular side is a small, distorted helix [transmembrane 2a (TM2a)], called the plug domain that is believed to impede the passage of water and solutes across the membrane. Access to the membrane from the water-filled hourglass-shaped interior of SecY is provided by the so-called lateral gate, formed by helices TM2b and TM7 (Fig. 1A). SecG is not required for function, but SecE is indispensable. Experimental (9–11) and computational studies (12–15) emphasize the importance of interactions of the gate helices with nascent-chain segments during TM helix insertion.Open in a separate windowFig. 1.Structure of P. furiosus SecYE and cartoon representation of the translocon-to-lipid bilayer partitioning of a TM helix. (A) Structure of SecYE in a lipid bilayer. The lipid headgroups are shown in ice-blue van der Waals representation. The 10 SecY helices are represented in gray cartoon format, except for TM2b (magenta) and TM7 (cyan). The SecE helices are colored green. Hydrophobic ring (HR) residues are drawn as yellow bonds. The plug domain TM2a is colored orange. (B) The apparent free energy of partitioning ΔG(z) of an alkyl sidechain (orange) with accessible surface area A_acc depends upon position z within the TM segment (solid red curve), because the atomic solvation parameter σ apparently depends upon position. Our results suggest an opposite behavior (dashed orange curve).The general belief is that nascent chains pass through the translocon via the normally closed HR, which must open in response to ribosome docking and nascent-chain elongation. When a TM segment passes through SecY, it is believed that the segment is shunted into the membrane through a simple partitioning process between water-filled SecY and the lipid environment (Fig. 1B). Hessa et al. (16, 17) characterized the translocon/membrane insertion energetics and established that the membrane insertion propensity depends on both the hydrophobicity and position of each residue within model TM segments (16, 17). Because the resulting “biological” hydrophobicity scale (16, 17) correlates strongly with physical hydrophobicity scales, translocon/membrane partitioning is believed to be similar to water/membrane partitioning of peptides (18), which is driven by the hydrophobic effect (19).The hallmark of hydrophobic partitioning is that the favorable partitioning free energy is linearly proportional to the solute nonpolar accessible surface area and is described by the atomic solvation parameter σ (20). For bulk water-to-hydrocarbon partitioning, the solvation parameter typically has a value of about −23 cal⋅mol⁻¹⋅Å⁻² (21). Öjemalm et al. (22) found that the apparent translocon-to-membrane free energy of insertion of nonproteinogenic amino acids at a particular position in the TM segment varied linearly with σ, as expected for hydrophobic-driven partitioning. However, σ was found to be less favorable than that for bulk water/hydrocarbon partitioning and to depend on position within the model TM segment (Fig. 1B). Near the ends of the segments, σ ≈ −6 cal⋅mol⁻¹⋅Å⁻², whereas in the center of the segment σ ≈ −10 cal⋅mol⁻¹⋅Å⁻² (22). These low values of σ caused us to examine the physical behavior of water within the translocon. Specifically, could the water properties within the translocon explain the magnitudes and position dependence of the translocon/bilayer partitioning solvation parameters? To address this question, we turned to atomistic molecular-dynamics (MD) simulations.From a number of crystallographic SecY structures (4–8), we chose the SecYE structure from Pyrococcus furiosus determined by Egea and Stroud (8), because it appears to be in a nearly open (“primed”) state as judged by the separation of the gate helices. We thought at the outset of our simulations that SecYE might close. However, SecYE remained stably open, which allowed close examination of the waters inside SecY. We found that waters deep within the translocon diffuse anomalously, have slow rotational dynamics, and have their dipoles aligned along the SecY axis. These properties indicate that translocons are not simple water-filled pores, which raises fundamental questions about the nature of translocon/bilayer partitioning of TM segments, and more generally about solute partitioning into compartments of restrained water molecules.     

Regulation and aggregation of intrinsically disordered peptides

Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer’s disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a “superposition of ensembles” hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.Most proteins in the human body have a well-defined, stable three-dimensional structure that is intimately tied to their physiological function. In the past few decades however, researchers have also identified a class of proteins that are natively unstructured. The latter, often referred to as intrinsically disordered proteins (IDPs) (1), are widespread and have the ability to quickly change their conformations to participate in a variety of biological processes. IDPs typically contain multiple charged side chains and few hydrophobic residues. Despite these characteristics, IDPs are not typically found in extended states but rather populate compact states due to hydrogen bonds and salt bridges (2, 3). IDP structures are highly regulated in the cell, and aberrant regulation often results in protein aggregation.In this paper we consider the effect of external agents, specifically osmolytes, in regulating IDP structure and aggregation properties. To carry out this study, we focused on the microtubule-associated protein tau, a soluble (4), archetypical IDP found in the nervous system that helps regulate and stabilize microtubules (5, 6). When the regulation of tau structure and activity is compromised, tau loses its ability to bind to microtubules, and disassociated tau proteins can aggregate into supramolecular assemblies with a cross-β structure (7–9) typical of amyloid fibers. This aggregation process is a pathological hallmark of Alzheimer’s disease and other forms of dementia known as tauopathies (10, 11). We consider here a segment of tau, the R2/wt fragment ₂₇₃GKVQIINKKLDL₂₈₄, which contains the highly aggregation prone paired helical filament (PHF6*) (VQIINK) region. R2/wt aggregates in vitro in a manner qualitatively similar to full-length tau, therefore we expect that the effects of osmolytes on R2/wt are similar to those on full-length tau.Our earlier work on this peptide showed that R2/wt is unstructured in solution, populating an ensemble of interconverting conformations (12). Two primary structural families emerged: a family consisting of extended conformations and a family consisting of compact conformations. The latter included structures that were stabilized by hydrogen bonding (primarily hairpins and, to a lesser extent, helices) or by salt bridges between aspartic acid (D) and lysine (K) groups, with salt bridges between K274 and D283 (located at opposite ends of the peptide) more prevalent than between adjacent residue pairs K280 and D283 or K281 and D283. Hydrogen bonded conformations are stabilized for enthalpic reasons, however, salt bridge ensembles are stabilized by both energetic and entropic contributions, as multiple conformations of the backbone are possible for any given salt bridge. The competition between salt bridge formation and hydrogen bonding is the reason that this peptide is intrinsically disordered, unable to find a unique structure. Conformations stabilized by hydrogen bonds are continually disrupted by the formation of salt bridges, and vice versa.Here, we propose a new hypothesis that we term “superposition of ensembles.” We propose that external regulating agents (osmolytes, crowders, etc.) or internal regulating mechanisms (mutations, posttranslational modifications, etc.) can shift the population of protein conformations, but do not necessarily introduce fundamentally new structures, and can thus define the basis of regulation. The subtle conformational shifts caused by these regulatory effects can then either enhance or suppress particular states, such as those possessing different levels of normal activity, or states with propensities to aggregate into abnormal pathological conformations. To explore this idea further, we consider the effect of the following external agents on the R2/wt tau conformation: (i) urea, an osmolyte used to denature globular proteins through presumed perturbations in peptide hydrogen bonding (13), and (ii) trimethylamine N-oxide (TMAO), an osmolyte that is known to stabilize globular proteins and act as a crowding agent (13). The effect of these agents on the monomeric state of R2/wt is studied using replica exchange molecular dynamics simulations, a methodology that enables efficient sampling of the diverse conformations populated by this peptide. The ability of R2/wt to aggregate in the presence of these external factors, in turn, is assayed experimentally using Thioflavin T (ThT) fluorescence spectroscopy to quantify aggregation rates and transmission electron microscopy (TEM) to determine aggregate morphologies. By combining statistics from molecular simulations with experimental observations, it becomes possible to study, at an atomic level, the intrinsic origins of protein disorder and regulation that play a leading role in most physiological processes.     

On the role of frustration in the energy landscapes of allosteric proteins

Natural protein domains must be sufficiently stable to fold but often need to be locally unstable to function. Overall, strong energetic conflicts are minimized in native states satisfying the principle of minimal frustration. Local violations of this principle open up possibilities to form the complex multifunnel energy landscapes needed for large-scale conformational changes. We survey the local frustration patterns of allosteric domains and show that the regions that reconfigure are often enriched in patches of highly frustrated interactions, consistent both with the idea that these locally frustrated regions may act as specific hinges or that proteins may "crack" in these locations. On the other hand, the symmetry of multimeric protein assemblies allows near degeneracy by reconfiguring while maintaining minimally frustrated interactions. We also anecdotally examine some specific examples of complex conformational changes and speculate on the role of frustration in the kinetics of allosteric change.     

Early events in the folding of four-helix-bundle heme proteins

Topologically homologous four-helix-bundle heme proteins exhibit striking diversity in their refolding kinetics. Cytochrome b562 has been reported to fold on a sub-millisecond time scale, whereas cytochrome c' refolding requires 10 s or more to complete. Heme dissociation in cytochrome b562 interferes with studies of folding kinetics, so a variant of cytochrome b562 (cytochrome c-b562) with a covalent c-type linkage to the heme has been expressed in Escherichia coli. Early events in the electron transfer-triggered folding of Fe(II)-cytochrome c-b562, along with those of Fe(II)-cytochrome c556, have been examined by using time-resolved absorption spectroscopy. Coordination of S(Met) to Fe(II) occurs within 10 mus after reduction of the denatured Fe(III)-cytochromes, and shortly thereafter (100 micros) the heme spectra are indistinguishable from those of the folded proteins. Under denaturing conditions, carbon monoxide binds to the Fe(II)-hemes in approximately 15 ms. By contrast, CO binding cannot compete with refolding in the Fe(II)-cytochromes, thereby confirming that the polypeptide encapsulates the heme in <10 ms. We suggest that Fe-S(Met) ligation facilitates refolding in these four-helix-bundle heme proteins by reducing the conformational freedom of the polypeptide chain.     

Enthalpy of helix-coil transition: missing link in rationalizing the thermodynamics of helix-forming propensities of the amino acid residues

It is known that different amino acid residues have effects on the thermodynamic stability of an alpha-helix. The underlying mechanism for the thermodynamic helical propensity is not well understood. The major accepted hypothesis is the difference in the side-chain configurational entropy loss upon helix formation. However, the changes in the side-chain configurational entropy explain only part of the thermodynamic helical propensity, thus implying that there must be a difference in the enthalpy of helix-coil transition for different residues. This work provides an experimental test to this hypothesis. Direct calorimetric measurements of folding of a model host peptide in which the helix formation is induced by metal binding is applied to a wide range of residue types, both naturally occurring and nonnatural, at the guest site. Based on the calorimetric results for 12 peptides, it was found that indeed there is a difference in the enthalpy of helix-coil transition for different amino acid residues, and simple empirical rules that define these differences are presented. The obtained difference in the enthalpies of helix-coil transition complement the differences in configurational entropies and provide the complete thermodynamic characterization of the helix-forming tendencies.     

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.     

Alpha-Helix folding in the presence of structural constraints

We have investigated the site-specific folding kinetics of a photoswitchable cross-linked alpha-helical peptide by using single (13)C = (18)O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6 degrees C), whereas at high temperatures (45 degrees C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained alpha-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an alpha-helical segment within a protein, rather than an isolated alpha-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.     

Structure of the complex between the cytosolic chaperonin CCT and phosducin-like protein

The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhLP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin.     

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.Recent years have seen a significant controversy arise regarding the behavior of the unfolded states of single-domain proteins in response to changing levels of chemical denaturant. That is, although small-angle X-ray scattering (SAXS) and single-molecule FRET (smFRET) results are all consistent with the argument that, at high levels of chemical denaturant (∼6 M or above), unfolded proteins adopt a swollen, self-avoiding ensemble well-approximated as an excluded volume random coil (1–3), the two approaches produce seemingly discrepant results for the dimensions of the unfolded states populated at lower (∼1 M) denaturant (4). For example, time-resolved SAXS studies of the unfolded state transiently populated when protein L is rapidly shifted from high guanidine hydrochloride (GuHCl) to low denaturant conditions produce no experimentally significant evidence for the compaction of this single-domain protein before folding (4, 5) [e.g., estimated radii of gyration of 25.1 ± 0.3 Å for the unfolded state at equilibrium in 7 M GuHCl and 24.9 ± 1.1 Å for the transient unfolded state populated at 0.67 M GuHCl; confidence intervals are SEs (4)]. In contrast, multiple smFRET studies conducted at equilibrium suggest that the unfolded state of dye-labeled protein L contracts by 20–40% over this same range of denaturant concentrations (6, 7) (Fig. 1). Moreover, this discrepancy seems to be nearly universal among single-domain proteins: whereas the results of at least a half-dozen SAXS studies on chemically unmodified, single-domain proteins fail to detect any experimentally significant evidence (at experimental precision of, typically, a few percent) of contraction (8–15), all of the more than one-dozen smFRET studies of dye-labeled, single-domain proteins reported to date have been interpreted in terms of unfolded states that contract as the denaturant concentration is lowered to ∼1 M (2, 6, 7, 16–26).Open in a separate windowFig. 1.Although it is widely accepted that chemically denatured proteins adopt an ensemble of conformations well-described as random coil (2), significant debate remains regarding the nature of the unfolded states populated at low denaturant concentrations (4). Specifically, time-resolved SAXS measurements collected within milliseconds of transfer from high to low denaturant suggest that chemically denatured single-domain proteins do not undergo any significant conformational change before folding itself (5, 12–14). (Left) Shown, for example, are SAXS profiles collected on protein L at equilibrium at 1 M (native conditions), at equilibrium at 4 M GuHCl (unfolding conditions), and transiently before folding on transfer to denaturant concentrations as low as 0.67 M; the near-superimposability of the scaled scattering data for the various unfolded states suggests that the unfolded chain does not change dimensions (to within experimental uncertainty of a few percent) before folding (4). (Right) The efficiency of smFRET observed across dye-labeled unfolded proteins, in contrast, invariably decreases significantly as the level of denaturant rises (6, 7, 16, 19, 20, 23), an observation that has universally been interpreted in terms of an unfolded state that expands significantly at higher denaturant concentrations. Shown are equilibrium smFRET data collected on dye-labeled protein L as a function of GuHCl concentration (6); the large shift in the placement of the peak corresponding to unfolded molecules (indicated by the blue bar) suggests that the chain contracts significantly when transferred from high to low denaturant. Fig. 1 (Left) reproduced with permission from ref. 4. Fig. 1 (Right) reproduced with permission from ref. 6.The discord between the views arising from SAXS and FRET presumably occurs because of some as yet unrecognized systematic or interpretational error associated with converting scattering profiles and/or observed transfer efficiencies into unfolded-state dimensions. Its exact origins, however, remain elusive. For example, although denaturant-dependent background scattering from the buffer must be subtracted to calculate molecular dimensions, native-state dimensions predicted from SAXS data are typically within experimental uncertainty of those calculated from crystal structures (27) and independent of denaturant concentration until the population of unfolded protein becomes significant (9). Similarly, although smFRET, by comparison, relies on several denaturant-dependent assumptions and approximations, no attempt to explain the discrepancy between smFRET and scattering as arising because of these assumptions and approximations has yet proven successful (4). Studies of rigid constructs, such as polyproline (23, 28–32), for example, indicate that the denaturant dependence of the index of refraction, the quantum yield, and the spectral shift of the dyes are all too small to account for the observed changes in transfer efficiency. Denaturant-dependent viscosity effects on the rate of conformational averaging have, likewise, been shown to be too small to account for the seeming inconsistency between SAXS- and FRET-based observations (33). Again, the source of this experimentally robust discrepancy remains stubbornly unclear (a more detailed discussion of this issue is in ref. 4).Key to the proper interpretation of any experiment is the availability of the appropriate controls. Because the pertinent SAXS experiments fail to produce evidence of unfolded-state contraction, the most relevant controls for these studies are positive controls establishing the technique’s ability to detect unfolded-state compaction, if it were to occur, of the magnitude seen by smFRET. Fortunately, these controls are already in hand: SAXS has repeatedly been shown to distinguish (at confidence intervals of many σ) between the dimensions of chemically unfolded, disulfide-free proteins, which generally coincide closely with expected random coil dimensions (2), and the 20–30% more compact (but still flexible and unfolded) states seen when the same chemically denatured protein is cross-linked through disulfide bonds (11, 12, 34, 35). Conversely, because smFRET studies of unfolded states are universally interpreted in terms of significant collapse, the most critical controls for smFRET would be negative controls (that is, the demonstration of the denaturant independence of smFRET across an unfolded polymer, the dimensions of which are known to be denaturant-independent). Here, for the first time to our knowledge, we report the results of just such a negative control. Specifically, we have performed smFRET and scattering studies of a flexible polymer known to adopt a conformation well-approximated as an expanded, excluded volume random walk (1–3) (herein referred to as a random coil) in aqueous solution across a wide range of conditions.