The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance to LeChatelier’s principle, that the unfolded state has smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔVu, has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔVu using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants which depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long range coupling intrinsic to the folded state. We also show that the energetics of the protein’s conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Delta+PHS. The temperature-dependent volumetric properties of the folded and unfolded states of the wild-type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level. We conclude that the expansivity of the unfolded state is, to a first approximation, temperature independent, while that of the folded state decreases with increasing temperature. Our data suggest that at low temperature the defining contribution to DeltaV comes mainly from excluded volume differences and DeltaV for unfolding is negative. In contrast, at high temperatures, differential solvation due to the increased exposed surface area of the unfolded state and, in particular, its larger thermal volume linked to the increased conformational dynamics of the unfolded state ensemble takes over and DeltaV for unfolding eventually becomes positive.

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.

The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

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The cover picture shows water molecules in different hydration shells at the surface of the model protein ubiquitin. The properties of proteins and the role of solvent in conformational dynamics is a central topic to the DFG-Foschergruppe (FOR 436) which forms the basis of this special issue. The polymorphism, dynamics and function of water at molecular interfaces is discussed with contributions from R.

peats, characteristic of the Repeats in ToXin (RTX) proteins, and an N-terminal proteolytic (passenger) domain. The exoprotease is secreted through a type I secretion system (TISS) composed of a tripartite complex formed by an ABC-transporter, a membrane fusion protein, and a TolC-like outer membrane protein. TISS substrates vary widely in size and function and include toxins, lipases, and proteases. The conserved feature of the substrate proteins is the presence of one or more RTX motifs. Previously, we have demonstrated that calcium regulates multiple conformations of AP. Calcium-induced RTX domain folding serves to chaperone the folding of the protease domain. Here we show that disruption of the calcium-binding sites alters both the affinity and cooperativity of calcium-induced folding, measured in the RTX domain and in the full-length protease, and that the binding sites are not isoenergetic. We have also evaluated the role of calcium in the secretion of the protease, as previous studies have suggested that calcium may facilitate protease secretion. Protein secretion was efficient when the passenger domain was maintained in an unfolded conformation and secreted into medium with high calcium concentrations. Secretion efficiencies decreased with mutations in the RTX domain and with passenger domains that were stable and folded in the bacterial cytoplasm. From these results we conclude calcium regulates protease conformation and may contribute to secretion efficiencies by maintaining specific protein conformations during translocation. These results provide a basis for understanding the calcium-associated secretion RTX proteins from multiple bacterial pathogens.