Folding Free Energy Surface Research Articles

Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.

Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.

TPP Riboswitch Populates Holo-Form-like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration.

Riboswitches are noncoding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate structures similar to the holo-form only in the presence of Mg2+ and absence of cognate ligand binding. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), the aptamer domain (AD) of TPP riboswitch undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into a structure similar to the crystal structure of the TPP holo-form even in the absence of the TPP ligand. The folding kinetics shows that TPP AD populates an intermediate due to the misalignment of two arms present in the structure, which acts as a kinetic trap, leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

Folding Free Energy Surfaces from Differential Scanning Calorimetry.

Protein folding/unfolding processes involve a large number of weak, non-covalent interactions and are more appropriately described in terms of the movement of a point representing protein conformation in a plot of internal free energy versus conformational degrees of freedom. While these energy landscapes have an astronomically large number of dimensions, it has been shown that many relevant aspects of protein folding can be understood in terms of their projections onto a few relevant coordinates. Remarkably, such low-dimensional free energy surfaces can be obtained from experimental DSC data using suitable analytical models. Here, we describe the experimental procedures to be used to obtain the high-quality DSC data that are required for free-energy surface analysis.

A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)1-8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in Ia and Ibp The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.

Mg2+ Sensing by an RNA Fragment: Role of Mg2+-Coordinated Water Molecules.

RNA molecules selectively bind to specific metal ions to populate their functional active states, making it important to understand their source of ion selectivity. In large RNA systems, metal ions interact with the RNA at multiple locations, making it difficult to decipher the precise role of ions in folding. To overcome this complexity, we studied the role of different metal ions (Mg2+, Ca2+, and K+) in the folding of a small RNA hairpin motif (5'-ucCAAAga-3') using unbiased all-atom molecular dynamics simulations. The advantage of studying this system is that it requires specific binding of a single metal ion to fold to its native state. We find that even for this small RNA, the folding free energy surface (FES) is multidimensional as different metal ions present in the solution can simultaneously facilitate folding. The FES shows that specific binding of a metal ion is indispensable for its folding. We further show that in addition to the negatively charged phosphate groups, the spatial organization of electronegative nucleobase atoms drives the site-specific binding of the metal ions. Even though the binding site cannot discriminate between different metal ions, RNA folds efficiently only in a Mg2+ solution. We show that the rigid network of Mg2+-coordinated water molecules facilitates the formation of important interactions in the transition state. The other metal ions such as K+ and Ca2+ cannot facilitate the formation of such interactions. These results allow us to hypothesize possible metal-sensing mechanisms in large metalloriboswitches and also provide useful insights into the design of appropriate collective variables for studying large RNA molecules using enhanced sampling methods.

Nonnative structure in a peptide model of the unfolded state of superoxide dismutase 1 (SOD1): Implications for ALS-linked aggregation

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding free-energy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using time-resolved FRET, we observed that the peptide corresponding to the Loop VII-β8 sequence at the SOD1 C terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state-like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered N terminus and the hydrophobic C terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.

Frustration and folding of a TIM barrel protein

Triosephosphate isomerase (TIM) barrel proteins have not only a conserved architecture that supports a myriad of enzymatic functions, but also a conserved folding mechanism that involves on- and off-pathway intermediates. Although experiments have proven to be invaluable in defining the folding free-energy surface, they provide only a limited understanding of the structures of the partially folded states that appear during folding. Coarse-grained simulations employing native centric models are capable of sampling the entire energy landscape of TIM barrels and offer the possibility of a molecular-level understanding of the readout from sequence to structure. We have combined sequence-sensitive native centric simulations with small-angle X-ray scattering and time-resolved Förster resonance energy transfer to monitor the formation of structure in an intermediate in the Sulfolobus solfataricus indole-3-glycerol phosphate synthase TIM barrel that appears within 50 μs and must at least partially unfold to achieve productive folding. Simulations reveal the presence of a major and 2 minor folding channels not detected in experiments. Frustration in folding, i.e., backtracking in native contacts, is observed in the major channel at the initial stage of folding, as well as late in folding in a minor channel before the appearance of the native conformation. Similarities in global and pairwise dimensions of the early intermediate, the formation of structure in the central region that spreads progressively toward each terminus, and a similar rate-limiting step in the closing of the β-barrel underscore the value of combining simulation and experiment to unravel complex folding mechanisms at the molecular level.

Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Atomic-Level Description of Protein Folding inside the GroEL Cavity.

Chaperonins (ubiquitous facilitators of protein folding) sequester misfolded proteins within an internal cavity, thus preventing protein aggregation during the process of refolding. GroEL, a tetradecameric bacterial chaperonin, is one of the most studied chaperonins, but the role of the internal cavity in the refolding process is still unclear. It has been suggested that rather than simply isolating proteins while they refold, the GroEL cavity actively promotes protein folding. A detailed characterization of the folding dynamics and thermodynamics of protein substrates encapsulated within the cavity, however, has been difficult to obtain by experimental means, due to the system's complexity and the many steps in the folding cycle. Here, we examine the influence of the GroEL cavity on protein folding based on the results of unbiased, atomistic molecular dynamics simulations. We first verified that the computational setup, which uses a recently developed state-of-the-art force field that more accurately reproduces the aggregation propensity of unfolded states, could recapitulate the essential structural dynamics of GroEL. In these simulations, the GroEL tetradecamer was highly dynamic, transitioning among states corresponding to most of the structures that have been observed experimentally. We then simulated a small, unfolded protein both in the GroEL cavity and in bulk solution and compared the protein's folding process within these two environments. Inside the GroEL cavity, the unfolded protein interacted strongly with the disordered residues in GroEL's C-terminal tails. These interactions stabilized the protein's unfolded states relative to its compact states and increased the roughness of its folding free-energy surface, resulting in slower folding compared to the rate in solution. For larger proteins, which are more typical GroEL substrates, we speculate that these interactions may allow substrates to more quickly escape kinetic traps associated with compact, misfolded states, thereby actively promoting folding.

Assessing and Manipulating Protein Folding Dynamics

While the folding energy landscape theory provides an overarching framework for understanding how proteins fold, a mechanistic elucidation of the folding mechanism of any specific protein still requires characterization of its conformational dynamics at the molecular level. However, this requirement amounts to a significant challenge to experimentalists as none of the existing techniques is capable of taking conformational snapshots along the folding pathway(s) with the necessary temporal and structural resolutions, let alone to directly and realistically determine the underlying folding free energy surface(s). In this talk, we will discuss several spectroscopic methods that could significantly improve our ability to assess the folding dynamics of specific structural elements in protein folding kinetic studies and, in some cases, to distinguish between different folding pathways. In addition, we will present examples showing how certain details of the folding free energy landscape of interest can be revealed by manipulating the corresponding folding dynamics via structural cross-linking.

Clusters of isoleucine, leucine, and valine side chains define cores of stability in high‐energy states of globular proteins: Sequence determinants of structure and stability

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.

Characterization of Folding Mechanisms of Trp-Cage and WW-Domain by Network Analysis of Simulations with a Hybrid-Resolution Model

In this study, we apply a hybrid-resolution model, namely, PACE, to characterize the free energy surfaces (FESs) of Trp-cage and a WW-domain variant along with the respective folding mechanisms. Unbiased, independent simulations with PACE are found to achieve together multiple folding and unfolding events for both proteins, allowing us to perform network analysis of the FESs to identify folding pathways. PACE reproduces for both proteins expected complexity hidden in the folding FESs, in particular metastable non-native intermediates. Pathway analysis shows that some of these intermediates are, actually, on-pathway folding intermediates and that intermediates kinetically closest to the native states can be either critical on-pathway or off-pathway intermediates, depending on the protein. Apart from general insights into folding, specific folding mechanisms of the proteins are resolved. We find that Trp-cage folds via a dominant pathway in which hydrophobic collapse occurs before the N-terminal helix forms; full incorporation of Trp6 into the hydrophobic core takes place as the last step of folding, which, however, may not be the rate-limiting step. For the WW-domain variant studied, we observe two main folding pathways with opposite orders of formation of the two hairpins involved in the structure; for either pathway, formation of hairpin 1 is more likely to be the rate-limiting step. Altogether, our results suggest that PACE combined with network analysis is a computationally efficient and valuable tool for the study of protein folding.

Folding free energy surfaces of three small proteins under crowding: validation of the postprocessing method by direct simulation

We have developed a ‘postprocessing’ method for modeling biochemical processes such as protein folding under crowded conditions (Qin and Zhou 2009 Biophys. J. 97 12–19). In contrast to the direct simulation approach, in which the protein undergoing folding is simulated along with crowders, the postprocessing method requires only the folding simulation without crowders. The influence of the crowders is then obtained by taking conformations from the crowder-free simulation and calculating the free energies of transferring to the crowders. This postprocessing yields the folding free energy surface of the protein under crowding. Here the postprocessing results for the folding of three small proteins under ‘repulsive’ crowding are validated by those obtained previously by the direct simulation approach (Mittal and Best 2010 Biophys. J. 98 315–20). This validation confirms the accuracy of the postprocessing approach and highlights its distinct advantages in modeling biochemical processes under cell-like crowded conditions, such as enabling an atomistic representation of the test proteins.

A Kinetic Zipper Model with Intrachain Interactions Applied to Nucleic Acid Hairpin Folding Kinetics

Single-stranded DNA and RNA hairpin structures with 4–10 nucleotides (nt) in the loop and 5–8 basepairs (bp) in the stem fold on 10–100 μs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ∼2-nt) is 10–100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔGnWC) and the rate at which a basepair is formed adjacent to an existing basepair (kbp+). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4–20-nt-long poly(dT) loops, with ΔGnWC ≈ −2.4 kcal/mol and kbp+ ≥ (1 ns)−1, in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.

Force-induced unfolding simulations of the human Notch1 negative regulatory region: possible roles of the heterodimerization domain in mechanosensing.

Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the γ-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling.

Estimation of protein folding free energy barriers from calorimetric data by multi-model Bayesian analysis

The realization that folding free energy barriers can be small enough to result in significant population of the species at the barrier top has sprouted in several methods to estimate folding barriers from equilibrium experiments. Some of these approaches are based on fitting the experimental thermogram measured by differential scanning calorimetry (DSC) to a one-dimensional representation of the folding free-energy surface (FES). Different physical models have been used to represent the FES: (1) a Landau quartic polynomial as a function of the total enthalpy, which acts as an order parameter; (2) the projection onto a structural order parameter (i.e. number of native residues or native contacts) of the free energy of all the conformations generated by Ising-like statistical mechanical models; and (3) mean-field models that define conformational entropy and stabilization energy as functions of a continuous local order parameter. The fundamental question that emerges is how can we obtain robust, model-independent estimates of the thermodynamic folding barrier from the analysis of DSC experiments. Here we address this issue by comparing the performance of various FES models in interpreting the thermogram of a protein with a marginal folding barrier. We chose the small α-helical protein PDD, which folds-unfolds in microseconds crossing a free energy barrier previously estimated as ~1 RT. The fits of the PDD thermogram to the various models and assumptions produce FES with a consistently small free energy barrier separating the folded and unfolded ensembles. However, the fits vary in quality as well as in the estimated barrier. Applying Bayesian probabilistic analysis we rank the fit performance using a statistically rigorous criterion that leads to a global estimate of the folding barrier and its precision, which for PDD is 1.3 ± 0.4 kJ mol(-1). This result confirms that PDD folds over a minor barrier consistent with the downhill folding regime. We have further validated the multi-model Bayesian approach through the analysis of two additional protein systems: gpW, a midsize single-domain with α + β topology that also folds in microseconds and has been previously catalogued as a downhill folder, and α-spectrin SH3, a domain of similar size but with a β-barrel fold, slow-folding kinetics and two-state-like thermodynamics. From a general viewpoint, the Bayesian analysis developed here results in a statistically robust, virtually model-independent, method to estimate the thermodynamic free-energy barriers to protein folding from DSC thermograms. Our method appears to be sufficiently accurate to consistently detect small differences in the barrier height, and thus opens up the possibility of characterizing experimentally the changes in thermodynamic folding barriers induced by single-point mutations on proteins within the downhill regime.

Integrated prediction of protein folding and unfolding rates from only size and structural class

Protein stability, folding and unfolding rates are all determined by the multidimensional folding free energy surface, which in turn is dictated by factors such as size, structure, and amino-acid sequence. Work over the last 15 years has highlighted the role of size and 3D structure in determining folding rates, resulting in many procedures for their prediction. In contrast, unfolding rates are thought to depend on sequence specifics and be much more difficult to predict. Here we introduce a minimalist physics-based model that computes one-dimensional folding free energy surfaces using the number of aminoacids (N) and the structural class (α-helical, all-β, or α-β) as only protein-specific input. In this model N sets the overall cost in conformational entropy and the net stabilization energy, whereas the structural class defines the partitioning of the stabilization energy between local and non-local interactions. To test its predictive power, we calibrated the model empirically and implemented it into an algorithm for the PREdiction of Folding and Unfolding Rates (PREFUR). We found that PREFUR predicts the absolute folding and unfolding rates of an experimental database of 52 proteins with accuracies of ±0.7 and ±1.4 orders of magnitude, respectively (relative to experimental spans of 6 and 8 orders of magnitude). Such prediction uncertainty for proteins vastly varying in size and structure is only two-fold larger than the differences in folding (±0.34) and unfolding rates (±0.7) caused by single-point mutations. Moreover, PREFUR predicts protein stability with an accuracy of ±6.3 kJ mol(-1), relative to the 5 kJ mol(-1) average perturbation induced by single-point mutations. The remarkable performance of our simplistic model demonstrates that size and structural class are the major determinants of the folding landscapes of natural proteins, whereas sequence variability only provides the final 10-20% tuning. PREFUR is thus a powerful bioinformatic tool for the prediction of folding properties and analysis of experimental data.

Improvements of network approach for analysis of the folding free‐energy surface of peptides and proteins

Folding network is an effective approach to investigate the high-dimensional free-energy surface of peptide and protein folding, and it can avoid the limitations of the projected free-energy surface based on two-order parameters. In this article, we present improvements of the effectiveness and accuracy of the folding network analysis based on Markov cluster (MCL) algorithm. We used this approach to investigate the folding free-energy surface of the beta-hairpin peptide trpzip2 and found the folding network is able to determine the basins and folding paths of trpzip2 more clearly and accurately than the two-dimensional free-energy surface.

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR⁎, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.

Zinc Binding Modulates the Entire Folding Free Energy Surface of Human Cu,Zn Superoxide Dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.