Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.     

The chaperonin cycle cannot substitute for prolyl isomerase activity, but GroEL alone promotes productive folding of a cyclophilin-sensitive substrate to a cyclophilin-resistant form.

The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.     

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.     

Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin.

The chaperonin GroEL binds unfolded polypeptides, preventing aggregation, and then mediates their folding in an ATP-dependent process. To understand the structural features in non-native polypeptides recognized by GroEL, we have used alpha-lactalbumin (alpha LA) as a model substrate. alpha LA (14.2 kDa) is stabilized by four disulfide bonds and a bound Ca2+ ion, offering the possibility of trapping partially folded disulfide intermediates between the native and the fully unfolded state. The conformers of alpha LA with high affinity for GroEL are compact, containing up to three disulfide bonds, and have significant secondary structure, but lack stable tertiary structure and expose hydrophobic surfaces. Complex formation requires almost the complete alpha LA sequence and is strongly dependent on salts that stabilize hydrophobic interactions. Unfolding of alpha LA to an extended state as well as the burial of hydrophobic surface upon formation of ordered tertiary structure prevent the binding to GroEL. Interestingly, GroEL interacts only with a specific subset of the many partially folded disulfide intermediates of alpha LA and thus may influence in vitro the kinetics of the folding pathways that lead to disulfide bonds with native combinations. We conclude that the chaperonin interacts with the hydrophobic surfaces exposed by proteins in a flexible compact intermediate or molten globule state.     

Mimicking the action of GroEL in molecular dynamics simulations: application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results support the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.     

Multi-scale simulations provide supporting evidence for the hypothesis of intramolecular protein translocation in GroEL/GroES complexes

The biological function of chaperone complexes is to assist the folding of non-native proteins. The widely studied GroEL chaperonin is a double-barreled complex that can trap non-native proteins in one of its two barrels. The ATP-driven binding of a GroES cap then results in a major structural change of the chamber where the substrate is trapped and initiates a refolding attempt. The two barrels operate anti-synchronously. The central region between the two barrels contains a high concentration of disordered protein chains, the role of which was thus far unclear. In this work we report a combination of atomistic and coarse-grained simulations that probe the structure and dynamics of the equatorial region of the GroEL/GroES chaperonin complex. Surprisingly, our simulations show that the equatorial region provides a translocation channel that will block the passage of folded proteins but allows the passage of secondary units with the diameter of an alpha-helix. We compute the free-energy barrier that has to be overcome during translocation and find that it can easily be crossed under the influence of thermal fluctuations. Hence, strongly non-native proteins can be squeezed like toothpaste from one barrel to the next where they will refold. Proteins that are already fairly close to the native state will not translocate but can refold in the chamber where they were trapped. Several experimental results are compatible with this scenario, and in the case of the experiments of Martin and Hartl, intra chaperonin translocation could explain why under physiological crowding conditions the chaperonin does not release the substrate protein.     

Structural plasticity and noncovalent substrate binding in the GroEL apical domain. A study using electrospay ionization mass spectrometry and fluorescence binding studies

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.     

GroEL–GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL–ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL–GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL–ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL–ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.     

Translocation boost protein-folding efficiency of double-barreled chaperonins

Incorrect folding of proteins in living cells may lead to malfunctioning of the cell machinery. To prevent such cellular disasters from happening, all cells contain molecular chaperones that assist nonnative proteins in folding into the correct native structure. One of the most studied chaperone complexes is the GroEL-GroES complex. The GroEL part has a "double-barrel" structure, which consists of two cylindrical chambers joined at the bottom in a symmetrical fashion. The hydrophobic rim of one of the GroEL chambers captures nonnative proteins. The GroES part acts as a lid that temporarily closes the filled chamber during the folding process. Several capture-folding-release cycles are required before the nonnative protein reaches its native state. Here we report molecular simulations that suggest that translocation of the nonnative protein through the equatorial plane of the complex boosts the efficiency of the chaperonin action. If the target protein is correctly folded after translocation, it is released. However, if it is still nonnative, it is likely to remain trapped in the second chamber, which then closes to start a reverse translocation process. This shuttling back and forth continues until the protein is correctly folded. Our model provides a natural explanation for the prevalence of double-barreled chaperonins. Moreover, we argue that internal folding is both more efficient and safer than a scenario where partially refolded proteins escape from the complex before being recaptured.     

Understanding the Folding-Function Tradeoff in Proteins

When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the “function-less” folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.     

Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Molecular chaperones are ATP‐consuming machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly. GroEL is an all‐purpose stochastic machine that assists misfolded substrate proteins to fold. The RNA chaperones such as CYT‐19, which are ATP‐consuming enzymes, help the folding of ribozymes that get trapped in metastable states for long times. GroEL does not interact with the folded proteins but CYT‐19 disrupts both the folded and misfolded ribozymes. The structures of GroEL and RNA chaperones are strikingly different. Despite these differences, the iterative annealing mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, T (apo), R (ATP bound), and R^″ (ADP bound). Analyses of the experimental data show that the efficiency of the GroEL–GroES machinery and mutants is determined by the resetting rate k _{R ″ → T} , which is largest for the wild‐type (WT) GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady‐state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.     

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops.     

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.     

Multiscale Coarse-Graining of the Protein Energy Landscape

A variety of coarse-grained (CG) models exists for simulation of proteins. An outstanding problem is the construction of a CG model with physically accurate conformational energetics rivaling all-atom force fields. In the present work, atomistic simulations of peptide folding and aggregation equilibria are force-matched using multiscale coarse-graining to develop and test a CG interaction potential of general utility for the simulation of proteins of arbitrary sequence. The reduced representation relies on multiple interaction sites to maintain the anisotropic packing and polarity of individual sidechains. CG energy landscapes computed from replica exchange simulations of the folding of Trpzip, Trp-cage and adenylate kinase resemble those of other reduced representations; non-native structures are observed with energies similar to those of the native state. The artifactual stabilization of misfolded states implies that non-native interactions play a deciding role in deviations from ideal funnel-like cooperative folding. The role of surface tension, backbone hydrogen bonding and the smooth pairwise CG landscape is discussed. Ab initio folding aside, the improved treatment of sidechain rotamers results in stability of the native state in constant temperature simulations of Trpzip, Trp-cage, and the open to closed conformational transition of adenylate kinase, illustrating the potential value of the CG force field for simulating protein complexes and transitions between well-defined structural states.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Sequential Steps in the Assembly of the Multimeric Outer Membrane Secretin PulD

Investigations into protein folding are largely dominated by studies on monomeric proteins. However, the transmembrane domain of an important group of membrane proteins is only formed upon multimerization. Here, we use in vitro translation-coupled folding and insertion into artificial liposomes to investigate kinetic steps in the assembly of one such protein, the outer membrane secretin PulD of the bacterial type II secretion system. Analysis of the folding kinetics, measured by the acquisition of distinct determinants of the native state, provides unprecedented evidence for a sequential multistep process initiated by membrane-driven oligomerization. The effects of varying the lipid composition of the liposomes indicate that PulD first forms a “prepore” structure that attains the native state via a conformational switch.