The role of negative selection in protein evolution revealed through the energetics of the native state ensemble

Knowing the determinants of conformational specificity is essential for understanding protein structure, stability, and fold evolution. To address this issue, a novel statistical measure of energetic compatibility between sequence and structure was developed using an experimentally validated model of the energetics of the native state ensemble. This approach successfully matched sequences from a diverse subset of the human proteome to their respective folds. Unexpectedly, significant energetic compatibility between ostensibly unrelated sequences and structures was also observed. Interrogation of these matches revealed a general framework for understanding the origins of conformational specificity within a proteome: specificity is a complex function of both the ability of a sequence to adopt folds other than the native, and ability of a fold to accommodate sequences other than the native. The regional variation in energetic compatibility indicates that the compatibility is dominated by incompatibility of sequence for alternative fold segments, suggesting that evolution of protein sequences has involved substantial negative selection, with certain segments serving as “gatekeepers” that presumably prevent alternative structures. Beyond these global trends, a size dependence exists in the degree to which the energetic compatibility is determined from negative selection, with smaller proteins displaying more negative selection. This partially explains how short sequences can adopt unique folds, despite the higher probability in shorter proteins for small numbers of mutations to increase compatibility with other folds. In providing evolutionary ground rules for the thermodynamic relationship between sequence and fold, this framework imparts valuable insight for rational design of unique folds or fold switches. Proteins 2016; 84:435–447. © 2016 Wiley Periodicals, Inc.     

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.     

Protein's native state stability in a chemically induced denaturation mechanism

In this work, we present a generalization of Zwanzig's protein unfolding analysis [Zwanzig, R., 1997. Two-state models of protein folding kinetics. Proc. Natl Acad. Sci. USA 94, 148-150; Zwanzig, R., 1995. Simple model of protein folding kinetics. Proc. Natl Acad. Sci. USA 92, 9801], in order to calculate the free energy change Delta(N)(D)F between the protein's native state N and its unfolded state D in a chemically induced denaturation. This Extended Zwanzig Model (EZM) is both based on an equilibrium statistical mechanics approach and the inclusion of experimental denaturation curves. It enables us to construct a suitable partition function Z and to derive an analytical formula for Delta(N)(D)F in terms of the number K of residues of the macromolecule, the average number nu of accessible states for each single amino acid and the concentration C(1/2) where the midpoint of the N<==>D transition occurs. The results of the EZM for proteins where chemical denaturation follows a sigmoidal-type profile, as it occurs for the case of the T70N human variant of lysozyme (PDB code: T70N) [Esposito, G., et al., 2003. J. Biol. Chem. 278, 25910-25918], can be splitted into two lines. First, EZM shows that for sigmoidal denaturation profiles, the internal degrees of freedom of the chain play an outstanding role in the stability of the native state. On the other hand, that under certain conditions DeltaF can be written as a quadratic polynomial on concentration C(1/2), i.e., DeltaF approximately aC(1/2)(2)+bC(1/2)+c, where a,b,c are constant coefficients directly linked to protein's size K and the averaged number of non-native conformations nu. Such functional form for DeltaF has been widely known to fit experimental measures in chemically induced protein denaturation [Yagi, M., et al., 2003. J. Biol. Chem. 278, 47009-47015; Asgeirsson, B., Guojonsdottir, K., 2006. Biochim. Biophys. Acta 1764, 190-198; Sharma, S., et al., 2006. Protein Pept. Lett. 13(4), 323-329; Salem, M., et al., 2006. Biochim. Biophys. Acta 1764(5), 903-912] so EZM can shed some light into the physical meaning of the experimental values for the a,b,c coefficients.     

High resolution approach to the native state ensemble kinetics and thermodynamics

Many biologically interesting functions such as allosteric switching or protein-ligand binding are determined by the kinetics and mechanisms of transitions between various conformational substates of the native basin of globular proteins. To advance our understanding of these processes, we constructed a two-dimensional free energy surface (FES) of the native basin of a small globular protein, Trp-cage. The corresponding order parameters were defined using two native substructures of Trp-cage. These calculations were based on extensive explicit water all-atom molecular dynamics simulations. Using the obtained two-dimensional FES, we studied the transition kinetics between two Trp-cage conformations, finding that switching process shows a borderline behavior between diffusive and weakly-activated dynamics. The transition is well-characterized kinetically as a biexponential process. We also introduced a new one-dimensional reaction coordinate for the conformational transition, finding reasonable qualitative agreement with the two-dimensional kinetics results. We investigated the distribution of all the 38 native nuclear magnetic resonance structures on the obtained FES, analyzing interactions that stabilize specific low-energy conformations. Finally, we constructed a FES for the same system but with simple dielectric model of water instead of explicit water, finding that the results were surprisingly similar in a small region centered on the native conformations. The dissimilarities between the explicit and implicit model on the larger-scale point to the important role of water in mediating interactions between amino acid residues.     

The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.     

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.     

Analysis of electrostatic interactions in the denatured state ensemble of the N-terminal domain of L9 under native conditions

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.     

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1

Information on the low-energy excited states of a given protein is important as this controls the structural adaptability and various biological functions of proteins such as co-operativity, response towards various external perturbations. In this article, we characterized individual residues in both non-myristoylated (non-myr) and myristoylated (myr) neuronal calcium sensor-1 (NCS-1) that access alternate states by measuring nonlinear temperature dependence of the backbone amide-proton (1H(N)) chemical shifts. We found that ~20% of the residues in the protein access alternative conformations in non-myr case, which increases to ~28% for myr NCS-1. These residues are spread over the entire polypeptide stretch and include the edges of α-helices and β-strands, flexible loop regions, and the Ca2(+)-binding loops. Besides, residues responsible for the absence of Ca2(+)-myristoyl switch are also found accessing alternative states. The C-terminal domain is more populated with these residues compared to its N-terminal counterpart. Individual EF-hands in NCS-1 show significantly different number of alternate states. This observation prompts us to conclude that this may lead to differences in their individual conformational flexibility and has implications on the functionality. Theoretical simulations reveal that these low-energy excited states are within an energy band of 2-4 kcal/mol with respect to the native state.     

Partial destabilization of native structure by a combination of heat and denaturant facilitates cold denaturation in a hyperthermophile protein

Cold denaturation is a phenomenon seen in many different proteins. However, there have been no reports so far of its occurrence in hyperthermophile proteins. Here, using a recombinant triosephosphate isomerase (PfuTIM) from the hyperthermophile archaeon, Pyrococcus furiosus, we show that the heating of this protein through the low temperature side of its thermal unfolding transition in the presence of guanidinium hydrochloride (GdmCl) results in the formation of partially-disordered conformational ensembles that retain considerable native-like secondary and tertiary structure. Unlike PfuTIM itself, these thermochemically obtained partially-disordered PfuTIM ensembles display cold denaturation as they are cooled to room temperature. The protein thus shows hysteresis, adopting different structural states in a manner dependent upon the nature of the heating and cooling treatment, rather than upon the initial and final conditions of temperature and GdmCl concentration, indicating that some sort of a kinetic effect influences structure adoption and retention. The structure lost through cooling of partially-disordered PfuTIM is found to be regained through heating. The ability of GdmCl to thus apparently destabilize the highly thermodynamically and kinetically stable structure of PfuTIM (sufficiently, to cause it to display observable cold-denaturation and heat-renaturation transitions, in real-time, with cooling and heating) offers support to current ideas concerning the how hyperthermophile proteins achieve their high kinetic stabilities, and suggests that desolvation-solvation barriers may be responsible for high kinetic stability.     

The native energy landscape for interleukin-1beta. Modulation of the population ensemble through native-state topology

A minimalist Go-model, with no energetic frustration in the native conformation, has been shown to describe accurately the folding pathway of the beta-trefoil protein, interleukin-1beta (IL-1beta). While it appears that these models successfully model transition states and intermediates between the unfolded and native ensembles, it is unclear how accurately they capture smaller, yet biologically relevant, structural changes within the native ensemble after energetic perturbation. Here, we address the following questions. Can a simple Go-model of interleukin-1beta, based on native topology, describe changes in structural properties of the native ensemble as the protein stability is changed? Or is it necessary to include a more explicit representation of atoms, electrostatic, hydrogen bonding, and van der Waals forces to describe these changes? The native ensemble of IL-1beta was characterized using a variety of experimental probes under native (0 M NaCl, guanidine hydrochloride (Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and in moderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear (1)H-(15)N nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) NMR spectra confirmed that the beta-trefoil global fold was largely intact under these three conditions. However, 25 of the 153 residues throughout the chain did demonstrate (13)C and (1)H-(15)N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl. Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) and destabilized (0.8 M Gdn-HCl) IL-1beta, no difference in steady-state (15)N-(1)H NOE enhancements were measured. Thus, the chemical shifts correlate with a global but limited increase in residue flexibility in the presence of Gdn-HCl. Minimalist simulations highlight the regions of greatest position shift between native and 0.8 M Gdn-HCl, which were determined experimentally. This correlation demonstrates that structural changes within the native ensemble of IL-1beta are, at least partially, governed by the principle of minimal energetic frustration.     

Multi-stage nature of the thermal denaturation process in collagen.

Thermal denaturation of acid-soluble collagen from polar cod (Eleginus gracialis) skin has been studied by scanning microcalorimetry and intrinsic spectrofluorimetry methods. The thermal denaturation process occurs in three independent stages reflecting the melting of 33 kDa, 97 kDa, and 230 kDa domains. Thermodynamical parameters of the collagen denaturation have been determined.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Heat and cold denaturation of beta-lactoglobulin B.

The thermal denaturation of bovine beta-lactoglobulin B was investigated by high-sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 20 mM phosphate buffer. The process was found to be a reversible, two-state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low-temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of beta-lactoglobulin B.     

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6 M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6 M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the protein's multiple functions.     

Mechanical denaturation of globular protein in the solid state

A method for taking stress-strain diagrams in microsamples prepared from glutaraldehyde-treated monocrystals and amorphous films of hen egg-white lysozyme has been developed. Analysis of the diagrams has shown that the deformation obeys Hooke's law within 0-2%. Upon further deformation of a crystalline sample (up to 6-10%), when a critical stress, sigma(cr), is reached, the protein molecules in the sample denature and become greatly extended. Depending on the crystal type and crystallographic direction, the sample length increases 2-4 times. The critical stress is essentially dependent on the factors affecting intra- and intermolecular interactions: temperature, hydration level and urea concentration. Mechanisms for mechanical denaturation are proposed.