$995,227
National Institute of Diabetes and Digestive and Kidney Diseases
Maryland
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. However, while the impact of pressure on the viscosity of water is well known, we find that elevated pressures increase the Rh of dioxane, a commonly used reference standard, and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction observed by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurements of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure and as a function of urea concentration show that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 angstrom, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) alpha-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of both unfolded ubiquitin and alpha-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions. Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses, such as heat shock or acidosis, HSP27 oligomers can dissociate into dimers and monomers, which leads to significantly enhanced chaperone activity. The structured core of the protein, the alpha-crystallin domain (ACD), forms dimers and can prevent the aggregation of substrate proteins to a similar degree as does the full-length protein. We have shown that when the ACD dimer dissociates into monomers, it partially unfolds and exhibits enhanced activity. Using solution-state NMR spectroscopy to characterize the structure and dynamics of the HSP27 ACD monomer, we have shown that the monomer is stabilized at low pH and that its backbone chemical shifts, N-15 relaxation rates, and 1H-15N residual dipolar couplings suggest structural changes and rapid motions in the region responsible for dimerization. By analyzing the solvent accessible and buried surface areas of sHSP structures in the context of a database of dimers that are known to dissociate into disordered monomers, we predict that ACD dimers from sHSPs across all kingdoms of life may partially unfold upon dissociation. This resulted in a general model in which conditional disorder-the partial unfolding of ACDs upon monomerization-is a common mechanism for sHSP activity. A series of novel three- and four-dimensional NMR experiments has been developed that provides access to the structure and dynamics of the 608-residue homodimeric Main protease of the SARS-CoV-2 virus. This protein is larger than what is readily accessible to the standard NMR approaches used, but the novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural analysis which reveals subtle but statistically very significant differences relative to all X-ray structures available so far. A characterization of the backbone dynamics shows a strong effect of ligands on the dynamics of the protein backbone in the active site region.