It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavitycontaining variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.energy landscape | fluorescence | volume change T he first observation that pressure unfolds proteins was made in 1914 by Bridgman (1). Despite numerous studies since then, the physical basis of the pressure-induced unfolding of proteins has not been explained. This difference in volume underlying pressure effects has been rationalized previously in terms of (i) increases in solvent density concomitant with solvation of exposed surfaces upon unfolding (2), (ii) modifications in the structure of bulk water leading to weakened hydrophobic interactions (3), and (iii) cavities in the folded state that are not present in the unfolded state (4-7). The goal of this study was to examine the structural origins of pressure unfolding of proteins. Twenty-five years ago Walter Kauzmann stressed the importance of understanding pressure effects (8): "Enthalpy and volume are equally fundamental properties of the (protein) unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behavior." He also noted important discrepancies between the volumetric properties of hydrophobic interactions and the pressure unfolding of proteins.To date, no conclusive explanation for the origins of pressure unfolding of proteins has been proposed. In the present work, 10 cavity-containing variants of staphylococcal nuclease (SNase) were engineered by substitution of internal core residues to Ala. Crystal structures of the variants were obtained to verify the exi...

The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ1–40 and Aβ1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ1–42 compared to that of Aβ1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ1–40 and Aβ1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ1–40 and Aβ1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings (3JHNHα, 3JC′C′, 3JC′Hα, 1JHαCα, 2JNCα, and 1JNCα) recorded for Aβ1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process.

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone 1H-15N, 15N-13C′, 1Hα-13Cα, and 13C′-13Cα one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with 1H-15N and 1Hα-13Cα RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in 13C′ chemical shift, 3JHNHα values, and 13Cα-13Cβ RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.