How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins.

Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self-avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N-terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10-58°C, with a Flory exponent, ν, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti-freeze protein (red-sfAFP) is unaffected by temperature and ionic strength and persists as a near-SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red-sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red-sfAFP had minimal effect on ν. Water remained a good solvent even with 21 consecutive glycine residues (ν > 0.5), and red-sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine-rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.

HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding.

To import large metabolites across the outer membrane of gram-negative bacteria, TonB-dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the Escherichia coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner-membrane proton motive force that powers transport [N. Noinaj, M. Guillier, T. J. Barnard, S. K. Buchanan, Annu. Rev. Microbiol. 64, 43­60 (2010)]. However, the role of TonB in rearranging the plug domain of BtuB to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding [S. J. Hickman, R. E. M. Cooper, L. Bellucci, E. Paci, D. J. Brockwell, Nat. Commun. 8, 14804 (2017)], while others propose force-independent pore formation by TonB binding [T. D. Nilaweera, D. A. Nyenhuis, D. S. Cafiso, eLife 10, e68548 (2021)], leading to breakage of a salt bridge termed the "Ionic Lock." Our hydrogen­deuterium exchange/mass spectrometry (HDX-MS) measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12-bound and the B12+TonB­bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region, with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate-binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

Engineered Metal-Binding Sites to Probe Protein Folding Transition States: Psi Analysis.

The formation of the transition state ensemble (TSE) represents the rate-limiting step in protein folding. The TSE is the least populated state on the pathway, and its characterization remains a challenge. Properties of the TSE can be inferred from the effects on folding and unfolding rates for various perturbations. A difficulty remains on how to translate these kinetic effects to structural properties of the TSE. Several factors can obscure the translation of point mutations in the frequently used method, "mutational Phi analysis." We take a complementary approach in "Psi analysis," employing rationally inserted metal binding sites designed to probe pairwise contacts in the TSE. These contacts can be confidently identified and used to construct structural models of the TSE. The method has been applied to multiple proteins and consistently produces a considerably more structured and native-like TSE than Phi analysis. This difference has significant implications to our understanding of protein folding mechanisms. Here we describe the application of the method and discuss how it can be used to study other conformational transitions such as binding.

Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or ß-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange.

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar.

Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative ß turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.

Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations.

The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol(-1) per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix-sheet = 0.5 kcal⋅mol(-1)), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR.

Benchmarking all-atom simulations using hydrogen exchange.

Long-time molecular dynamics (MD) simulations are now able to fold small proteins reversibly to their native structures [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. These results indicate that modern force fields can reproduce the energy surface near the native structure. To test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability and H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2° structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations.

Context and force field dependence of the loss of protein backbone entropy upon folding using realistic denatured and native state ensembles.

The loss of conformational entropy is the largest unfavorable quantity affecting a protein's stability. We calculate the reduction in the number of backbone conformations upon folding using the distribution of backbone dihedral angles (ϕ,ψ) obtained from an experimentally validated denatured state model, along with all-atom simulations for both the denatured and native states. The average loss of entropy per residue is TΔS(BB)(U-N) = 0.7, 0.9, or 1.1 kcal·mol(-1) at T = 298 K, depending on the force field used, with a 0.6 kcal·mol(-1) dispersion across the sequence. The average equates to a decrease of a factor of 3-7 in the number of conformations available per residue (f = Ω(Denatured)/Ω(Native)) or to a total of f(tot) = 3(n)-7(n) for an n residue protein. Our value is smaller than most previous estimates where f = 7-20, that is, our computed TΔS(BB)(U-N) is smaller by 10-100 kcal mol(-1) for n = 100. The differences emerge from our use of realistic native and denatured state ensembles as well as from the inclusion of accurate local sequence preferences, neighbor effects, and correlated motions (vibrations), in contrast to some previous studies that invoke gross assumptions about the entropy in either or both states. We find that the loss of entropy primarily depends on the local environment and less on properties of the native state, with the exception of α-helical residues in some force fields.

A "Link-Psi" strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable.

Using a combined crosslinking-ψ analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two ß strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the ψ value at another region (ψ identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in ψ values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and ψ-analysis for examining potential transition state heterogeneity in globular proteins.

Psi-constrained simulations of protein folding transition states: implications for calculating.

Psi-analysis has been used to identify interresidue contacts in the transition state ensemble (TSE) of ubiquitin and other proteins. The magnitude of psi depends on the degree to which an inserted bihistidine (biHis) metal ion binding site is formed in the TSE. A psi equal to zero or one indicates that the biHis site is absent or fully native-like, respectively, while a fractional psi implies that in the TSE, the biHis site recovers only part of the binding-induced stabilization of the native state. All-atom Langevin dynamics simulations of the TSE are performed with restrictions imposed only on the distances between the pairs of residues with experimentally determined psi of unity. When a site with a fractional psi lies adjacent to a site with psi = 1, the fractional psi generally signifies that the "fractional site" has a distorted geometry in the TSE. When a fractional site is distal to the sites with psi = 1, however, the histidines sample configurations in which the site is absent. The simulations indicate that the psi = 1 sites by themselves can be used to generate a well-defined TSE having near-native topology. values calculated from the TS simulations exhibit mixed agreement with the experimental values. The origin and implication of the disparities are discussed.

Quantifying the structural requirements of the folding transition state of protein A and other systems.

The B-domain of protein A is a small three-helix bundle that has been the subject of considerable experimental and theoretical investigation. Nevertheless, a unified view of the structure of the transition-state ensemble (TSE) is still lacking. To characterize the TSE of this surprisingly challenging protein, we apply a combination of psi analysis (which probes the role of specific side-chain to side-chain contacts) and kinetic H/D amide isotope effects (which measures hydrogen-bond content), building upon previous studies using mutational phi analysis (which probes the energetic influence of side-chain substitutions). The second helix is folded in the TSE, while helix formation appears just at the carboxy and amino termini of the first and third helices, respectively. The experimental data suggest a homogenous yet plastic TS with a native-like topology. This study generalizes our earlier conclusion, based on two larger alpha/beta proteins, that the TSEs of most small proteins achieve approximately 70% of their native state's relative contact order. This high percentage limits the degree of possible TS heterogeneity and requires a reevaluation of the structural content of the TSE of other proteins, especially when they are characterized as small or polarized.