Low force unfolding of a single-domain protein by parallel pathways

Deviations from linearity in the dependence of the logarithm of protein unfolding rates, log ku(f), as a function of mechanical force, f, measurable in single molecule experiments, can arise for many reasons. In particular, upward curvature in log ku(f) as a function of f implies that the underlying energy landscape must be multidimensional with the possibility that unfolding ensues by parallel pathways. Here, simulations using the SOP-SC model of a wild type β-sandwich protein and several mutants, with immunoglobulin folds, show upward curvature in the unfolding kinetics. There are substantial changes in the structures of the transition state ensembles as force is increased, signaling a switch in the unfolding pathways. Our results, when combined with previous theoretical and experimental studies, show that parallel unfolding of structurally unrelated single domain proteins can be determined from the dependence of log ku(f) as a function of force (or log ku[C] where [C] is the denaturant concentration).

Dissecting the statistical properties of the linear extrapolation method of determining protein stability

The linear extrapolation method to determine protein stability from denaturant-induced unfolding experiments is based on the observation that the free energy of unfolding is often a linear function of the denaturant concentration. The value in the absence of denaturant is then estimated by extrapolation from this linear relationship. Parameters and their confidence intervals are typically estimated by nonlinear least-squares regression. We have compared different methods for calculating confidence intervals and found that a simple method based on linear theory gives accurate results. We have also compared three different parameterizations of the linear extrapolation method and show that the most commonly used form is problematic since the stability and m-value are correlated in the nonlinear least-squares analysis. Parameter correlation can in some cases causes problems in the estimation of confidence intervals and regions and should be avoided when possible. Two alternative parameterizations show much less correlation between parameters.

Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.

Direct and Indirect Models for Protein Chemical Denaturation are Characteristic of Opposite Dynamic Properties

Two major models, namely direct and indirect models, have been proposed for the protein chemical denaturation but it remains challenging to experimentally demonstrate and distinguish between them. Here, by use of CD and NMR spectroscopy, we succeeded in differentiating the effects on a small but well-folded protein WW4, of GdmCl and NaSCN at diluted concentrations (≥200 mM). Both denaturants up to 200 mM have no alternation of its average structure but do reduce its thermodynamic stability to different degrees. Despite acting as the stronger denaturant, GdmCl only weakly interacts with amide protons, while NaSCN shows extensive interactions with both hydrophobic side chains and amide protons. Although both denaturants show no significant perturbation on overall ps-ns backbone dynamics of WW4, GdmCl suppresses while NaSCN enhances its μs-ms backbone dynamics in a denaturant concentration dependent manner. Quantitative analysis reveals that although they dramatically raise exchange rates, GdmCl slightly increases while NaSCN reduces the population of the major conformational state. Our study represents the first report deciphering that GdmCl and NaSCN appear to destabilize a protein following two models respectively, which are characteristic of opposite μs-ms dynamics.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.