Cooperative Folding Research Articles

Using a Coarse-Grained Modeling Framework to Identify Oligomeric Motifs with Tunable Secondary Structure.

Coarse-grained modeling can be used to explore general theories that are independent of specific chemical detail. In this paper, we present cg_openmm, a Python-based simulation framework for modeling coarse-grained hetero-oligomers and screening them for structural and thermodynamic characteristics of cooperative secondary structures. cg_openmm facilitates the building of coarse-grained topology and random starting configurations, setup of GPU-accelerated replica exchange molecular dynamics simulations with the OpenMM software package, and features a suite of postprocessing thermodynamic and structural analysis tools. In particular, native contact analysis, heat capacity calculations, and free energy of folding calculations are used to identify and characterize cooperative folding transitions and stable secondary structures. In this work, we demonstrate the capabilities of cg_openmm on a simple 1-1 Lennard-Jones coarse-grained model, in which each residue contains 1 backbone and 1 side-chain bead. By scanning both nonbonded and bonded force-field parameter spaces at the coarse-grained level, we identify and characterize sets of parameters which result in the formation of stable helices through cooperative folding transitions. Moreover, we show that the geometries and stabilities of these helices can be tuned by manipulating the force-field parameters.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

Probing Pressure Effects on Core Packing of a Repeat Protein using 13C-1H NMR

Repeat proteins greatly simplify the identification of the sequence determinants for protein folding cooperativity. We use the leucine rich repeat (LRR) domain of the tumor suppressor pp32 as a model. It is composed of five LRR with a capping domain on each of its termini. Previous 15N-1H high-pressure HSQC experiments revealed that intermediates are populated in the folding of pp32, and in several cavity-containing variants. In order to obtain more information about these intermediates, in particular about the core packing, we collected 13C-1H HSQC spectra as a function of pressure on three cavity-containing variants of pp32, one in the N-terminus (I7A), one in the C-terminus (L139A) and one in the central core (L60A).

Programming folding cooperativity of the dimeric i-motif with DNA frameworks for sensing small pH variations.

The response sensitivity of a molecular sensor is determined by the folding cooperativity of its responsive module. Using an H+-responsive dimeric DNA i-motif as a model, we demonstrate the enhancement of its folding cooperativity through preorganization by a DNA framework, and with it we fabricate robust intracellular pH sensors with high response sensitivity.

Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase.

The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in the folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to 'U-shaped' DPO4 folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4-DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, which is a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.

Increased supraorganization of respiratory complexes is a dynamic multistep remodelling in response to proteostasis stress.

Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard the mitochondrial proteome and mitochondrial functions despite increased aggregation during proteasome inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supraorganization of respiratory complexes (RCs) in proteasome-inhibited cells that occurs simultaneously with increased pelletable aggregation of RC subunits inside mitochondria. Complex II (CII) and complex V (CV) subunits are increasingly incorporated into oligomers. Complex I (CI), complex III (CIII) and complex IV (CIV) subunits are engaged in supercomplex formation. We unravel unique quinary states of supercomplexes during early proteostatic stress that exhibit plasticity and inequivalence of constituent RCs. The core stoichiometry of CI and CIII is preserved, whereas the composition of CIV varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC subunits into holocomplexes and supercomplexes re-establishes steady-state stoichiometry. Overall, the mechanism of increased supraorganization of RCs mimics the cooperative unfolding and folding pathways for protein folding, but is restricted to RCs and is not observed for any other mitochondrial protein complexes.This article has an associated First Person interview with the first author of the paper.

Lessons about Protein Folding and Binding from Archetypal Folds.

The function of proteins as biological nanomachines relies on their ability to fold into complex 3D structures, bind selectively to partners, and undergo conformational changes on cue. The native functional structures, and the rates of interconversion between conformational states (folded-unfolded, bound-free), are all encoded in the physical chemistry of their amino acid sequence. However, despite extensive research over decades, this code has proven difficult to fully crack, in terms of both prediction and understanding the molecular mechanisms at play.Earlier work on single-domain proteins reported a commonality of slow rates (10-2-102 s-1) and simple behavior in both kinetic and thermodynamic unfolding experiments, which suggested the process was all-or-none and thereby analogous to a chemical reaction (e.g., A ⇄ B). In the absence of a first-principles pre-exponential factor for protein (un)folding dynamics, the rates could only be interpreted in relative terms, e.g., the changes induced by mutation, and hence, neither the height of nor the entropic contribution to the free energy barriers was known. The rates were also many orders of magnitude too slow for direct atomistic simulations, and the computational focus was on predicting rate changes induced by mutation via coarse grained simulations. However, even the effects of mutation proved to be strikingly homogeneous with all experimental data clustering at ∼1/3 of the free energy perturbation recovered on folding and ∼2/3 on unfolding.The implementation of ultrafast kinetic methods turned the field upside down because they allowed researchers to measure the time scales of elementary (un)folding motions, which set the pre-exponential factor for protein conformational transitions at ∼1 μs. In parallel, we and others set out to investigate the simplest possible protein structures capable of autonomous folding, which we defined as archetypal folds. The rationale was to recapitulate the hierarchical organization of protein structure, starting from the bottom up. The study of fold archetypes ended up opening new research avenues in protein (un)folding, but also making unexpected connections with the folding upon binding of intrinsically disordered proteins and suggesting their functioning as conformational rheostats.This Account describes our work on the kinetic, thermodynamic, mechanistic, and functional analysis of fold archetypes. We first discuss the kinetic studies, emphasizing their impact on our understanding of (un)folding rates, of barrierless (downhill) folding, and as benchmarks for atomistic simulations. We continue with the thermodynamic analysis, introducing the differential scanning calorimetry, multiprobe, and NMR approaches that we developed to dissect their gradual, minimally cooperative (un)folding transitions and to probe the underlying mechanisms with unprecedented detail. The last two sections cover single-molecule analyses and some recent, mostly computational, results on the exploration of possible biological and technological roles for the gradual conformational transitions of fold archetypes.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

The Cold-Unfolded State Is Expanded but Contains Long- and Medium-Range Contacts and Is Poorly Described by Homopolymer Models.

Cold unfolding of proteins is predicted by the Gibbs-Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium- and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.

Cotranslational folding cooperativity of contiguous domains of α-spectrin

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

Design of Calcium-Binding Proteins to Sense Calcium.

Calcium controls numerous biological processes by interacting with different classes of calcium binding proteins (CaBP’s), with different affinities, metal selectivities, kinetics, and calcium dependent conformational changes. Due to the diverse coordination chemistry of calcium, and complexity associated with protein folding and binding cooperativity, the rational design of CaBP’s was anticipated to present multiple challenges. In this paper we will first discuss applications of statistical analysis of calcium binding sites in proteins and subsequent development of algorithms to predict and identify calcium binding proteins. Next, we report efforts to identify key determinants for calcium binding affinity, cooperativity and calcium dependent conformational changes using grafting and protein design. Finally, we report recent advances in designing protein calcium sensors to capture calcium dynamics in various cellular environments.

Assembly of a DNA Origami Chinese Knot by Only 15% of the Staple Strands.

As a giant leap in DNA self-assembly, DNA origami has exhibited an unprecedented ability to construct nanostructures with arbitrary shapes and sizes. In typical DNA origami, hundreds of short DNA staple strands fold a long, single-stranded (ss) DNA scaffold cooperatively into designed nanostructures. However, large numbers of DNA strands are expensive and would hinder applications such as pharmaceutical investigations because of the complicated components. Therefore, one challenge is how to reduce the number of staple strands needed to construct DNA origami. For a DNA origami structure, the scale-free folding pattern of the scaffold strand is determined by staple strands at the branching vertexes. Simple duplex regions help to define the size-related features of the origami geometry. In this study, we hypothesized that a scaffold strand can be correctly folded into a designed topology by using only staple strands involved in branching vertexes. After assembly, any remaining, flexible, single-stranded regions of the scaffold could be converted into rigid duplexes by DNA polymerase to achieve the designed geometric structures. To demonstrate the concept, we used only 18 staple strands (covering 15 % of the scaffold strand) to assemble a porous DNA nanostructure, which was visualized by atomic force microscopy (AFM). This study helps understanding of the role of cooperativity in origami folding, and provides a cost-effective approach for small-scale prototyping DNA origami.

Molecular Dynamics of Villin Headpiece-36 Under Different Hydration Levels

Hydrophobic cores are sites of complex protein folding and misfolding patterns, and they often define protein stability and folding overall. A protein subdomain of the protein villin, headpiece-36 (HP36) is a convenient model system to study the hydrophobic core interactions and dynamics due to its small size (36 residues) and cooperative folding. Here, we study the molecular dynamics of HP36 in the solid powder state, with the focus on the dynamics of three phenylalanine sidechains, F47, F51, and F58 belonging to the hydrophobic core under different (100%, 40%, and 10%) hydration levels. The potential mean force (PMF) of the sidechain rotations of these phenylalanine residues are computed using umbrella sampling, providing implications on the plasticity and rigidity of hydrophobic core dynamics, which are compared with experimental solid-state NMR results.

Confinement and Crowding Effects on Folding of a Multidomain Y-Family DNA Polymerase.

Proteins in vivo endure highly various interactions from the luxuriant surrounding macromolecular cosolutes. Confinement and macromolecular crowding are the two major effects that should be considered while comparing the results of protein dynamics from in vitro to in vivo. However, efforts have been largely focused on single domain protein folding up to now, and the quantifications of the in vivo effects in terms of confinements and crowders on modulating the structure and dynamics as well as the physical understanding of the underlying mechanisms on multidomain protein folding are still challenging. Here we developed a topology-based model to investigate folding of a multidomain Y-family DNA polymerase (DPO4) within spherical confined space and in the presence of repulsive and attractive crowders. We uncovered that the entropic component of the thermodynamic driving force led by confinements and repulsive crowders increases the stability of folded states relative to the folding intermediates and unfolded states, while the enthalpic component of the thermodynamic driving force led by attractive crowders gives rise to the opposite effects with less stability. We found that the shapes of DPO4 conformations influenced by the confinements and the crowders are quite different even when only the entropic component of the thermodynamic driving force is considered. We uncovered that under all in vivo conditions, the folding cooperativity of DPO4 decreases compared to that in bulk. We showed that the loss of folding cooperativity can promote the sequential domain-wise folding, which was widely found in cotranslational multidomain protein folding, and effectively prohibit the backtracking led by topological frustrations during multidomain protein folding processes.

Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) ~20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.

Enthalpically-driven ligand recognition and cavity solvation of bovine odorant binding protein

Lipocalins are a widely distributed family of extracellular proteins typically involved in the transport of small hydrophobic molecules. To gain new insights into the molecular basis that governs ligand recognition by this ancient protein family, the binding properties of the domain-swapped dimer bovine odorant binding protein (bOBP) and its monomeric mutant bOBP121G+ were characterized using calorimetric techniques and molecular dynamics simulations. Thermal unfolding profiles revealed that the isolated bOBP subunits behave as a cooperative folding unit. In addition, bOBP and bOBP121G+ exhibited similar ligand binding properties, characterized by a non-classical hydrophobic effect signature. The energetic differences in the binding of bOBP to 1-hexen-3-ol and the physiological ligand 1-octen-3-ol were strikingly larger than those observed for the interaction of other lipocalins with congeneric ligands. MD simulations revealed that the recurrent opening of transient pores in the submicrosecond timescale allows a profuse exchange of water molecules between the protein interior and the surrounding solvent. This picture contrasts with other lipocalins whose ligand-free binding cavities are devoid of solvent molecules. Furthermore, the simulations indicated that internal water molecules solvate the protein cavity suboptimally, forming fewer hydrogen bonds and having lower density and higher potential energy than bulk water molecules. Upon ligand occupation, water molecules were displaced from the binding cavity in an amount that depended on the ligand size. Taken together, calorimetric and MD-simulation results are consistent with a significant contribution of cavity desolvation to the enthalpically-driven interaction of bOBP with its hydrophobic ligands.

Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5' and 3' ends of the cloverleaf core, and these extensions get trimmed before addition of the 3'-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5' and 3' ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

Improving folding properties of computationally designed proteins.

While the field of computational protein design has witnessed amazing progression in recent years, folding properties still constitute a significant barrier towards designing new and larger proteins. In order to assess and improve folding properties of designed proteins, we have developed a genetics-based folding assay and selection system based on the essential enzyme, orotate phosphoribosyl transferase from Escherichia coli. This system allows for both screening of candidate designs with good folding properties and genetic selection of improved designs. Thus, we identified single amino acid substitutions in two failed designs that rescued poorly folding and unstable proteins. Furthermore, when these substitutions were transferred into a well-structured design featuring a complex folding profile, the resulting protein exhibited native-like cooperative folding with significantly improved stability. In protein design, a single amino acid can make the difference between folding and misfolding, and this approach provides a useful new platform to identify and improve candidate designs.

On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations

Single-molecule force spectroscopy has proven extremely beneficial in elucidating folding pathways for membrane proteins. Here, we simulate these measurements, conducting hundreds of unfolding trajectories using our fast Upside algorithm for slow enough speeds to reproduce key experimental features that may be missed using all-atom methods. The speed also enables us to determine the logarithmic dependence of pulling velocities on the rupture levels to better compare to experimental values. For simulations of atomic force microscope measurements in which force is applied vertically to the C-terminus of bacteriorhodopsin, we reproduce the major experimental features including even the back-and-forth unfolding of single helical turns. When pulling laterally on GlpG to mimic the experiment, we observe quite different behavior depending on the stiffness of the spring. With a soft spring, as used in the experimental studies with magnetic tweezers, the force remains nearly constant after the initial unfolding event, and a few pathways and a high degree of cooperativity are observed in both the experiment and simulation. With a stiff spring, however, the force drops to near zero after each major unfolding event, and numerous intermediates are observed along a wide variety of pathways. Hence, the mode of force application significantly alters the perception of the folding landscape, including the number of intermediates and the degree of folding cooperativity, important issues that should be considered when designing experiments and interpreting unfolding data.

Cellular Concentrations of Nucleotide Diphosphate-Chelated Magnesium Ions Accelerate Catalysis by RNA and DNA Enzymes.

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg2+free. We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.