Cooperative Folding Research Articles

Spatial ranges of driving forces are a key determinant of protein folding cooperativity and rate diversity

The physical basis of two-state-like folding transitions and the tremendous diversity in folding rates is elucidated by directly simulating the folding kinetics of 52 representative proteins. Relative to the results from a common modeling approach, the diversity of the simulated folding rates can be increased from ~10(2.1) to the experimental ~10(6.0) by a modest decrease in the spatial range of the attractive potential. The required theoretical range is consistent with desolvation physics and is notably much more permissive than that needed for two-state-like homopolymer collapse.

Sketching protein aggregation with a physics-based toy model

We explore the applicability of a single-bead coarse-grained molecular model to describe the competition between protein folding and aggregation. We have designed very simple and regular sequences, based on our previous studies on peptide aggregation, that successfully fold into the three main protein structural families (all-α, all-β, and α + β). Thanks to equilibrium computer simulations, we evaluate how temperature and concentration promote aggregation. Aggregates have been obtained for all the amino acid sequences considered, showing that this process is common to all proteins, as previously stated. However, each structural family presents particular characteristics that can be related to its specific balance between hydrogen bond and hydrophobic interactions. The model is very simple and has limitations, yet it is able to reproduce both the cooperative folding of isolated polypeptide chains with regular sequences and the formation of different types of aggregates at high concentrations.

Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of Binary, Ternary, and Quaternary DNA Complexes

We report herein the structuring of single-stranded thymine-rich DNA sequences into peptide-DNA hairpin triplex structures via designed melamine-thymine nucleobase recognition. Melamine-displaying α-peptides were synthesized with the general form (EM*)n, where M* denotes a lysine residue side chain derivatized with melamine, a bifacial hydrogen bond complement for thymine. We have found that (EM*)n peptides, which we term bifacial peptide nucleic acid (bPNA), function as a noncovalent template for thymine-rich DNA tracts. Unstructured DNA of the general form dTnCmTn are bound to (EM*)n peptides and fold into cooperatively melting 1:1 bPNA-DNA hairpin complexes with dissociation constants in the submicromolar to low nanomolar range for n = 4-10. As the length of the interface (n) is decreased, the melting temperature of the bPNA-DNA complex drops significantly, though Kd increases are less substantial, suggestive of strong enthalpy-entropy compensation. This is borne out by differential scanning calorimetry analysis, which indicates enthalpically driven bPNA-DNA base-stacking that becomes markedly less exothermic as the recognition surface n decreases in size. The recognition interface tolerates a high number of "mismatches" and indicates half-site, or monofacial, recognition between melamine and thymine may occur if only 1 complementary nucleobase is available. Association correlates directly with fractional thymine content, with optimal binding when the number of T-T sites match the number of melamine units. Interestingly, when a DNA host has more T-T sites than melamine sites on bPNA, two or three bPNAs can bind to a single DNA, resulting in ternary and quaternary complexes that have higher thermal stability than the binary (1:1) bPNA-DNA complex, suggestive of cooperative multisite binding. In contrast, when two bPNAs of different lengths bind to the same DNA host, a ternary complex is formed with two melting transitions, corresponding to independent melting of each bPNA component from the complex. These data demonstrate that melamine-displaying bPNA recognize thymine-rich DNA in predictable and multifaceted ways that allow binding affinity, structure stability, and stoichiometry to be tuned through simple bPNA length modification and matching with DNA length. Synthetic bPNA structuring elements may be useful tools for biotechnology.

The interplay between protein stability and dynamics in conformational diseases: The case of hPGK1 deficiency

Conformational diseases often show defective protein folding efficiency in vivo upon mutation, affecting protein properties such as thermodynamic stability and folding/unfolding/misfolding kinetics as well as the interactions of the protein with the protein homeostasis network. Human phosphoglycerate kinase 1 (hPGK1) deficiency is a rare inherited disease caused by mutations in hPGK1 that lead to loss-of-function. This disease offers an excellent opportunity to explore the complex relationships between protein stability and dynamics because of the different unfolding mechanisms displayed towards chemical and thermal denaturation. This work explores these relationships using two thermostable mutants (p.E252A and p.T378P) causing hPGK1 deficiency and WT hPGK1 using proteolysis and chemical denaturation. p.T378P is degraded ~30-fold faster at low protease concentrations (here, the proteolysis step is rate-limiting) and ~3-fold faster at high protease concentrations (where unfolding kinetics is rate-limiting) than WT and p.E252A, indicating that p.T378P is thermodynamically and kinetically destabilized. Urea denaturation studies support the decrease in thermodynamic stability and folding cooperativity for p.T378P, as well as changes in folding/unfolding kinetics. The present study reveals changes in the folding landscape of hPGK1 upon mutation that may affect protein folding efficiency and stability in vivo, also suggesting that native state stabilizers and protein homeostasis modulators may help to correct folding defects in hPGK1 deficiency. Moreover, detailed kinetic proteolysis studies are shown to be powerful and simple tools to provide deep insight into mutational effects on protein folding and stability in conformational diseases.

Cooperative Unfolding of Compact Conformations of the Intrinsically Disordered Protein Osteopontin

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.

Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein.

A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two "primitive" versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment.

Quantifying the Drive-Response Relationships between Residues in Protein Folding

In protein folding processes, mutual correlation and cooperativity are commonly used to describe the interactions between different residues in a protein. However, these metrics do not provide any information on the causality relationships between these residues. The drive-response relationships in protein folding processes are still poorly studied and, due to technical limitations, are difficult to measure experimentally. The information theory transfer entropy (TE) provides a direct measurement of causality between two times series. We have used this method to quantify the causality between residues in the folding processes generated from molecular dynamics simulations for six small proteins, i.e., Trp-cage, BBA, Villin, WW domain, NTL9 and BBL (Lindorff-Larsen K. et al. (2011) Science. 334, 517-520). Instead of using one single TE value, we calculated the time-dependent TE using the residue-based Q-scores along the folding/unfolding processes. Preliminary analysis reveals that the residues in the hydrophobic cores are frequently involved in drive-response relationships, indicating the critical roles of these residues. The time-dependent TE also allows us to identify key events during the folding and evaluate the contributions from non-native contacts. We expect further studies may disclose more details on the causality relationships of the residues and the folding mechanisms of these proteins.

Calcium-Induced Folding and Secretion of Alkaline Protease (Apra) from Pseudomonas Aeruginosa

Alkaline protease (AP) is a known virulence factor secreted from Pseudomonas aeruginosa (Pa), which causes serious infection in patients with cystic fibrosis, cancer and severe burns. AP consists of two domains: a C-terminal calcium-binding domain containing glycine- and aspartate nonapeptide repeats, characteristic of the Repeats in ToXin (RTX) proteins, and an N-terminal proteolytic (passenger) domain. The exoprotease is secreted through a type I secretion system (TISS) composed of a tripartite complex formed by an ABC-transporter, a membrane fusion protein, and a TolC-like outer membrane protein. TISS substrates vary widely in size and function and include toxins, lipases, and proteases. The conserved feature of the substrate proteins is the presence of one or more RTX motifs. Previously, we have demonstrated that calcium regulates multiple conformations of AP. Calcium-induced RTX domain folding serves to chaperone the folding of the protease domain. Here we show that disruption of the calcium-binding sites alters both the affinity and cooperativity of calcium-induced folding, measured in the RTX domain and in the full-length protease, and that the binding sites are not iso-energetic. We have also evaluated the role of calcium in the secretion of the protease, as previous studies have suggested that calcium may facilitate protease secretion. Protein secretion was efficient when the passenger domain was maintained in an unfolded conformation and secreted into medium with high calcium concentrations. Secretion efficiencies decreased with mutations in the RTX domain and with passenger domains that were stable and folded in the bacterial cytoplasm. From these results we conclude calcium regulates protease conformation and may contribute to secretion efficiencies by maintaining specific protein conformations during translocation. These results provide a basis for understanding the calcium-associated secretion RTX proteins from multiple bacterial pathogens.

Patterned recognition of amines and ammonium ions by a stimuli-responsive foldamer-based hexameric oligophenol host

A stimuli-responsive hexameric oligophenol host undergoes amine-induced co-operative folding from a more fluorescent, more linear structure into less fluorescent, more curved or helically folded states, enabling easy identification and classification of the bound amine guests.

Transition paths, diffusive processes, and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.

Decrease in RNA Folding Cooperativity by Deliberate Population of Intermediates in RNA G‐Quadruplexes

Keeping a broad (RNA) perspective: conventional biochemical detection systems only have a 100-fold linear response range. The range of potassium concentrations detected by an RNA G-quadruplex sequence can be broadened by intentionally populating multiple intermediate folding states. The folding of the RNA G-quadruplexes was monitored by both circular dichroism and intrinsic fluorescence spectroscopy.

Decrease in RNA Folding Cooperativity by Deliberate Population of Intermediates in RNA G-Quadruplexes

Decrease in RNA Folding Cooperativity by Deliberate Population of Intermediates in RNA G-Quadruplexes

Truncation of a β-Barrel Scaffold Dissociates Intrinsic Stability from Its Propensity to Aggregation

Δ98Δ is a functional all-β sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, Δ98Δ exhibits a nativelike β-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and Δ98Δ regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.

Modulation of Folding Kinetics of Repeat Proteins: Interplay between Intra- and Interdomain Interactions

Repeat proteins have unique elongated structures that, unlike globular proteins, are quite modular. Despite their simple one-dimensional structure, repeat proteins exhibit intricate folding behavior with a complexity similar to that of globular proteins. Therefore, repeat proteins allow one to quantify fundamental aspects of the biophysics of protein folding. One important feature of repeat proteins is the interfaces between the repeating units. In particular, the distribution of stabilities within and between the repeats was previously suggested to affect their folding characteristics. In this study, we explore how the interface affects folding kinetics and cooperativity by investigating two families of repeat proteins, namely, the Ankyrin and tetratricopeptide repeat proteins, which differ in the number of interfacial contacts that are formed between their units as well as in their folding behavior. By using simple topology-based models, we show that modulating the energetic strength of the interface relative to that of the repeat itself can drastically change the protein stability, folding rate, and cooperativity. By further dissecting the interfacial contacts into several subsets, we isolated the effects of each of these groups on folding kinetics. Our study highlights the importance of interface connectivity in determining the folding behavior.

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

Thermodynamic Dissection of the Intrinsically Disordered N-terminal Domain of Human Glucocorticoid Receptor

Intrinsically disordered (ID) sequence segments are abundant in cell signaling proteins and transcription factors. Because ID regions commonly fold as part of their intracellular function, it is crucial to understand the folded states as well as the transitions between the unfolded and folded states. Specifically, it is important to determine 1) whether large ID segments contain different thermodynamically and/or functionally distinct regions, 2) whether any ID regions fold upon activation, 3) the degree of coupling between the different ID regions, and 4) whether the stability of ID domains is a determinant of function. In this study, we thermodynamically characterized the full-length ID N-terminal domain (NTD) of human glucocorticoid receptor (GR) and two of its naturally occurring translational isoforms. The protective osmolyte trimethylamine N-oxide (TMAO) was used to induce folding transitions. Each of the three NTD isoforms was found to undergo a cooperative folding transition that is thermodynamically indistinguishable (based on m-values) from that of a globular protein of similar size. The extrapolated stabilities for the NTD isoforms showed clear correlation with the known activities of their corresponding GR translational isoforms. The data reveal that the full-length NTD can be viewed as having at least two thermodynamically coupled regions, a functional region, which is indispensable for GR transcriptional activity, and a regulatory region, the length of which serves to regulate the stability of NTD and thus the activity of GR. These results suggest a new functional paradigm whereby steroid hormone receptors in particular and ID proteins in general can have multiple functionally distinct ID regions that interact and modulate the stability of important functional sites.

Canonical and micro-canonical analysis of folding of trpzip2: An all-atom replica exchange Monte Carlo simulation study

The density of states of trpzip2, a β-hairpin peptide, has been explored at all-atom level. Replica exchange Monte Carlo method was used for sufficient sampling over a wide range of temperature. Micro-canonical analysis was performed to confirm that the phase transition behavior of this two-state folder is first-order-like. Canonical analysis of heat capacity suggests that hydrogen bonding interaction exerts a considerable positive influence on folding cooperativity, in contrast, hydrophobic interaction is insufficient for high degree of folding cooperativity. Furthermore, we explain physical nature of the folding process from free energy landscape perspective and extensively analyse hydrogen bonding and stacking energy.

Multiple molecule effects on the cooperativity of protein folding transitions in simulations

Though molecular simulation of proteins has made notable contributions to the study of protein folding and kinetics, disagreement between simulation and experiment still exists. One of the criticisms levied against simulation is its failure to reproduce cooperative protein folding transitions. This weakness has been attributed to many factors such as a lack of polarizability and adequate capturing of solvent effects. This work, however, investigates how increasing the number of proteins simulated simultaneously can affect the cooperativity of folding transitions--a topic that has received little attention previously. Two proteins are studied in this work: phage T4 lysozyme (Protein Data Bank (PDB) ID: 7LZM) and phage 434 repressor (PDB ID: 1R69). The results show that increasing the number of proteins molecules simulated simultaneously leads to an increase in the macroscopic cooperativity for transitions that are inherently cooperative on the molecular level but has little effect on the cooperativity of other transitions. Taken as a whole, the results identify one area of consideration to improving simulations of protein folding.

A thermodynamic definition of protein domains

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.