Local Stability and Dynamics of Apocytochrome b562 Examined by the Dependence of Hydrogen Exchange on Hydrostatic Pressure,

The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models.foldons ͉ PPOE model ͉ staphylococcal nuclease T hat proteins might fold by way of some predetermined pathway was first suggested by C. Levinthal (1), who reasoned that folding through a random search process would take far longer than the subsecond time scale often observed. Experimental work driven by this concept found evidence for distinct pathway intermediates (2). However, theoretical work showed that the energetically downhill nature of the conformational search might by itself be sufficient to drive random folding on a realistic time scale (3). No specific pathway would then be necessary. This scenario has been formalized in the funneled energy landscape view for protein folding (4-8). Following this precedent, experimental results for a number of proteins have been similarly interpreted in terms of multiple unrelated parallel pathways (9)(10)(11)(12)(13)(14)(15)(16). The critical observation is that protein folding can be kinetically heterogeneous. Different fractions of a folding population fold at different rates and exhibit different populated intermediates, suggesting alternative pathways. This view is often represented in terms of multiple tracks through the funneled energy landscape. We refer to this view as the independent unrelated pathways (IUP) model to emphasize that intermediate structures in the different tracks have no particular relationship.A much more determinate pathway is supported by structural information derived experimentally for folding intermediates in many proteins (17)(18)(19)(20). This information indicates that protein folding intermediates are definitively native-like, constructed from cooperative foldon units of the target native protein. It appears that native-like foldon units are formed and docked into place one after another in a stepwise sequence, each one guided and stabilized by pr...

Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Bédard

Mallela

Mayne

et al. 2008

“…In line with this, several residues located in this C-terminal cooperative unit made greater contributions to ΔV u . Cooperative folding units have been explored in several proteins, including cytochrome c (65) and apocytochrome b 562 (66), using different perturbing agents.…”

Section: Discussionmentioning

confidence: 99%

A hypothesis to reconcile the physical and chemical unfolding of proteins

Oliveira

Silva

2015

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein-solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though proteinurea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p 1/2 and larger ΔV u ); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.protein folding | hydrostatic pressure | urea | NMR | SAXS

“…Because the mechanism of destabilization of the various perturbations (e.g., chemical denaturants, pH, temperature, pressure) likely varies, it is advantageous to use a single denaturing perturbation (although see ref. 49).…”

Section: Discussionmentioning

confidence: 99%

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

Nucci

Fuglestad

Athanasoula

et al. 2014

Self Cite

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T 4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation