Mechanism of chaperone function in small heat-shock proteins. Fluorescence studies of the conformations of T4 lysozyme bound to alphaB-crystallin

To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind alpha-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimane-labeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of alpha-crystallin to T4L. This directly demonstrates the two-mode nature of the binding process by the alpha-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where alpha-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.     

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.     

Comparison between denaturant- and temperature-induced unfolding pathways of protein: a lattice Monte Carlo simulation

Denaturant-induced unfolding of protein is simulated by using a Monte Carlo simulation with a lattice model for protein and denaturant. Following the binding theory for denaturant-induced unfolding, the denaturant molecules are modeled to interact with protein by nearest-neighbor interactions. By analyzing the conformational states on the unfolding pathway of protein, the denaturant-induced unfolding pathway is compared with the temperature-induced unfolding pathway under the same condition; that is, the free energies of unfolding under two different pathways are equal. The two unfoldings show markedly different conformational distributions in unfolded states. From the calculation of the free energy of protein as a function of the number fraction (Q0) of native contacts relative to the total number of contacts, it is found that the free energy of the largely unfolded state corresponding to low Q0 (0.1 < Q0 < 0.5) under temperature-induced unfolding is lower than that under denaturant-induced unfolding, whereas the free energy of the unfolded state close to the native state (Q0 > 0.5) is lower in denaturant-induced unfolding than in temperature-induced unfolding. A comparison of two unfolding pathways reveals that the denaturant-induced unfolding shows a wider conformational distribution than the temperature-induced unfolding, while the temperature-induced unfolding shows a more compact unfolded state than the denaturant-induced unfolding especially in the low Q0 region (0.1 < Q0 < 0.5).     

Mechanism of a hereditary cataract phenotype. Mutations in alphaA-crystallin activate substrate binding

We present a novel hypothesis for the molecular mechanism of autosomal dominant cataract linked to two mutations in the alphaA-crystallin gene of the ocular lens. AlphaA-crystallin is a molecular chaperone that plays a critical role in the suppression of protein aggregation and hence in the long term maintenance of lens optical properties. Using a steady state binding assay in which the chaperone-substrate complex is directly detected, we demonstrate that the mutations result in a substantial increase in the level of binding to non-native states of the model substrate T4 lysozyme. The structural basis of the enhanced binding is investigated through equivalent substitutions in the homologous heat shock protein 27. The mutations shift the oligomeric equilibrium toward a dissociated multimeric form previously shown to be the binding-competent state. In the context of a recent thermodynamic model of chaperone function that proposes the coupling of small heat shock protein activation to the substrate folding equilibrium (Shashidharamurthy, R., Koteiche, H. A., Dong, J., and McHaourab, H. S. (2005) J. Biol. Chem. 280, 5281-5289), the enhanced binding by the alphaA-crystallin mutants is predicted to shift the substrate folding equilibrium toward non-native intermediates, i.e. the mutants promote substrate unfolding. Given the high concentration of alphaA-crystallin in the lens, the molecular basis of pathogenesis implied by our results is a gain of function that leads to the binding of undamaged proteins and subsequent precipitation of the saturated alpha-crystallin complexes in the developing lens of affected individuals.     

Mechanism of chaperone function in small heat-shock proteins. Phosphorylation-induced activation of two-mode binding in alphaB-crystallin

The consequences of alphaB-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alphaB-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively destabilized proteins having similar structures in the folded state. alphaB-crystallin and its variants differentially recognize the T4L mutants, binding the more destabilized ones to a larger extent. Furthermore, the aspartate substitutions result in an increase in the extent of binding to the same T4L mutant and in the appearance of biphasic binding isotherms. The latter indicates the presence of two modes of binding characterized by different affinities and different numbers of binding sites. The transition to two-mode binding can also be induced by temperature or pH activation of the second mode. The similarity between the phosphorylation, pH, and temperature effects suggests a common structural origin. The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.     

Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy

Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.     

Binding of destabilized betaB2-crystallin mutants to alpha-crystallin: the role of a folding intermediate

Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to individual crystallins. One type of attractive force involves the recognition by alpha-crystallins of modified proteins prone to unfolding and aggregation. In this paper, we explore the energetic threshold and the structural determinants for the formation of a stable complex between alpha-crystallin and betaB2-crystallin as a consequence of destabilizing mutations in the latter. The mutations were designed in the framework of a folding model that proposes the equilibrium population of a monomeric intermediate. Binding to alpha-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in betaB2-crystallin. alpha-Crystallin binds the various betaB2-crystallin mutants, although with a significantly lower affinity relative to destabilized T4 lysozyme mutants. The extent of binding, while reflective of the overall destabilization, is determined by the dynamic population of a folding intermediate. The existence of the intermediate is inferred from the biphasic bimane emission unfolding curve of a mutant designed to disrupt interactions at the dimer interface. The results of this paper are consistent with a model in which the interaction of alpha-crystallins with substrates is not solely triggered by an energetic threshold but also by the population of excited states even under favorable folding conditions. The ability of alpha-crystallin to detect subtle changes in the population of betaB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.     

Geofold: topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding.     

Folding and stability of mutant scaffolding proteins defective in P22 capsid assembly.

Bacteriophage P22 scaffolding subunits are elongated molecules that interact through their C termini with coat subunits to direct icosahedral capsid assembly. The soluble state of the subunit exhibits a partially folded intermediate during equilibrium unfolding experiments, whose C-terminal domain is unfolded (Greene, B., and King, J. (1999) J. Biol. Chem. 274, 16135-16140). Four mutant scaffolding proteins exhibiting temperature-sensitive defects in different stages of particle assembly were purified. The purified mutant proteins adopted a similar conformation to wild type, but all were destabilized with respect to wild type. Analysis of the thermal melting transitions showed that the mutants S242F and Y214W further destabilized the C-terminal domain, whereas substitutions near the N terminus either destabilized a different domain or affected interactions between domains. Two mutant proteins carried an additional cysteine residue, which formed disulfide cross-links but did not affect the denaturation transition. These mutants differed both from temperature-sensitive folding mutants found in other P22 structural proteins and from the thermolabile temperature-sensitive mutants described for T4 lysozyme. The results suggest that the defects in these mutants are due to destabilization of domains affecting the weak subunit-subunit interactions important in the assembly and function of the virus precursor shell.     

Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

Equilibrium unfolding studies of monellin: the double-chain variant appears to be more stable than the single-chain variant

To improve our understanding of the contributions of different stabilizing interactions to protein stability, including that of residual structure in the unfolded state, the small sweet protein monellin has been studied in both its two variant forms, the naturally occurring double-chain variant (dcMN) and the artificially created single-chain variant (scMN). Equilibrium guanidine hydrochloride-induced unfolding studies at pH 7 show that the standard free energy of unfolding, ΔG°(U), of dcMN to unfolded chains A and B and its dependence on guanidine hydrochloride (GdnHCl) concentration are both independent of protein concentration, while the midpoint of unfolding has an exponential dependence on protein concentration. Hence, the unfolding of dcMN like that of scMN can be described as two-state unfolding. The free energy of dissociation, ΔG°(d), of the two free chains, A and B, from dcMN, as measured by equilibrium binding studies, is significantly lower than ΔG°(U), apparently because of the presence of residual structure in free chain B. The value of ΔG°(U), at the standard concentration of 1 M, is found to be ～5.5 kcal mol(-1) higher for dcMN than for scMN in the range from pH 4 to 9, over which unfolding appears to be two-state. Hence, dcMN appears to be more stable than scMN. It seems that unfolded scMN is stabilized by residual structure that is absent in unfolded dcMN and/or that native scMN is destabilized by strain that is relieved in native dcMN. The value of ΔG°(U) for both protein variants decreases with an increase in pH from 4 to 9, apparently because of the thermodynamic coupling of unfolding to the protonation of a buried carboxylate side chain whose pK(a) shifts from 4.5 in the unfolded state to 9 in the native state. Finally, it is shown that although the thermodynamic stabilities of dcMN and scMN are very different, their kinetic stabilities with respect to unfolding in GdnHCl are very similar.     

Towards a consistent modeling of protein thermodynamic and kinetic cooperativity: how applicable is the transition state picture to folding and unfolding?

To what extent do general features of folding/unfolding kinetics of small globular proteins follow from their thermodynamic properties? To address this question, we investigate a new simplified protein chain model that embodies a cooperative interplay between local conformational preferences and hydrophobic burial. The present four-helix-bundle 55mer model exhibits protein-like calorimetric two-state cooperativity. It rationalizes native-state hydrogen exchange observations. Our analysis indicates that a coherent, self-consistent physical account of both the thermodynamic and kinetic properties of the model leads naturally to the concept of a native state ensemble that encompasses considerable conformational fluctuations. Such a multiple-conformation native state is seen to involve conformational states similar to those revealed by native-state hydrogen exchange. Many of these conformational states are predicted to lie below native baselines commonly used in interpreting calorimetric data. Folding and unfolding kinetics are studied under a range of intrachain interaction strengths as in experimental chevron plots. Kinetically determined transition midpoints match well with their thermodynamic counterparts. Kinetic relaxations are found to be essentially single-exponential over an extended range of model interaction strengths. This includes the entire unfolding regime and a significant part of a folding regime with a chevron rollover, as has been observed for real proteins that fold with non-two-state kinetics. The transition state picture of protein folding and unfolding is evaluated by comparing thermodynamic free energy profiles with actual kinetic rates. These analyses suggest that some chevron rollovers may arise from an internal frictional effect that increasingly impedes chain motions with more native conditions, rather than being caused by discrete deadtime folding intermediates or shifts of the transition state peak as previously posited.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway

Intermediates along a protein's folding pathway can play an important role in its biology. Previous kinetics studies have revealed an early folding intermediate for T4 lysozyme, a small, well-characterized protein composed of an N-terminal and a C-terminal subdomain. Pulse-labeling hydrogen exchange studies suggest that residues from both subdomains contribute to the structure of this intermediate. On the other hand, equilibrium native state hydrogen experiments have revealed a high-energy, partially unfolded form of the protein that has an unstructured N-terminal subdomain and a structured C-terminal subdomain. To resolve this discrepancy between kinetics and equilibrium data, we performed detailed kinetics analyses of the folding and unfolding pathways of T4 lysozyme, as well as several point mutants and large-scale variants. The data support the argument for the presence of two distinct intermediates, one present on each side of the rate-limiting transition state barrier. The effects of circular permutation and site-specific mutations in the wild-type and circular permutant background, as well as a fragment containing just the C-terminal subdomain, support a model for the unfolding intermediate with an unfolded N-terminal and a folded C-terminal subdomain. Our results suggest that the partially unfolded form identified by native state hydrogen exchange resides on the folded side of the rate-limiting transition state and is, therefore, under most conditions, a "hidden" intermediate.     

Thermodynamic stability of the C-terminal domain of the human inducible heat shock protein 70

The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.     

Equilbrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (II).

The reversible unfolding of alpha-lactalbumin by guanidine hydrochloride, was studied at 25.0 degrees C in a relatively low concentration range of the denaturant (0.80-2.00 mol/l) by means of difference spectra and pH-jump measurements. The unfolding was shown to occur between two states, N and D, because apparent rate-constants of the unfolding and the refolding reactions depended only on pH. All curves plotted as the logarithmical equilibrium constant log KD against pH could fall on the same base curve by shifting each curve along the log KD axis. From the dependence of the logarithmic rate constant on pH, master curves could also be made for the forward and the backward reactions. The dependence of these master curves on pH indicates that the groups affecting the pH dependence of the unfolding are three residues with pKN = 3.3 and pKA = pKD = 4.4, one residue with pKN = pKA = 3.8 and pKD = 4.4, and one residue with pKN = 5.8 and pKA = pKD = 6.3, where A indicates the activated state. On the other hand, from the denaturant activity dependence of the shift factors required for making the master curves, the value of the intrinsic binding constant of the denaturant to the protein was found to be similar to that obtained from previous measurements at pH 5.5. Differences between the numbers of the binding sites of the denaturant on the denaturated and the native proteins, and between those on the activated and the native proteins were shown to be 5.3 and 2.1, respectively. The free energy of stabilization in the native-like environment also shows that the protein in the native state is more unstable than lysozyme.     

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.     

How cytochrome c folds, and why: submolecular foldon units and their stepwise sequential stabilization

Native state hydrogen exchange experiments have shown that the cytochrome c (Cyt c) protein consists of five cooperative folding-unfolding units, called foldons. These are named, in the order of increasing unfolding free energy, the nested-Yellow, Red, Yellow, Green, and Blue foldons. Previous results suggest that these units unfold in a stepwise sequential way so that each higher energy partially unfolded form includes all of the previously unfolded lower free energy units. If this is so, then selectively destabilizing any given foldon should equally destabilize each subsequent unfolding step above it in the unfolding ladder but leave the lower ones before it unaffected. To perform this test, we introduced the mutation Glu62Gly, which deletes a salt link in the Yellow unit and destabilizes the protein by 0.8 kcal/mol. Native state hydrogen exchange and other experiments show that the stability of the Yellow unit and the states above it in the free energy ladder are destabilized by about the same amount while the lower lying states are unaffected. These results help to confirm the sequential stepwise nature of the Cyt c unfolding pathway and therefore a similar refolding pathway. The steps in the pathway are dictated by the concerted folding-unfolding property of the individual unit foldons; the order of steps is determined by the sequential stabilization of progressively added foldons in the native context. Much related information for Cyt c strongly conforms with this mechanism. Its generality is supported by available information for other proteins.     

In silico profiling of tyrosine kinases binding specificity and drug resistance using Monte Carlo simulations with the ensembles of protein kinase crystal structures

The molecular basis of the tyrosine kinases binding specificity and drug resistance against cancer drugs Imatinib and Dasatinib is elucidated using Monte Carlo simulations of the inhibitor-receptor binding with the ensembles of protein kinase crystal structures. In silico proteomics analysis unravels mechanisms by which structural plasticity of the tyrosine kinases is linked with the conformational preferences of Imatinib and Dasatinib in achieving effective drug binding with a distinct spectrum of the tyrosine kinome. The differences in the inhibitor sensitivities to the ABL kinase mutants are rationalized based on variations in the binding free energy profiles with the conformational states of the ABL kinase. While Imatinib binding is highly sensitive to the activation state of the enzyme, the computed binding profile of Dasatinib is remarkably tolerant to the conformational state of ABL. A comparative analysis of the inhibitor binding profiles with the clinically important ABL kinase mutants has revealed an excellent agreement with the biochemical and proteomics data. We have found that conformational adaptability of the kinase inhibitors to structurally different conformational states of the tyrosine kinases may have pharmacological relevance in acquiring a specific array of potent activities and regulating a scope of the inhibitor resistance mutations. This study outlines a useful approach for understanding and predicting the molecular basis of the inhibitor sensitivity against potential kinase targets and drug resistance.