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Structural and dynamic characterization of a denatured state ensemble under non-denaturing conditions
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Structural and dynamic characterization of a denatured state ensemble under non-denaturing conditions
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Structural and dynamic characterization of a denatured state ensemble under non-denaturing conditions
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Dissertation
Structural and dynamic characterization of a denatured state ensemble under non-denaturing conditions
2006
Overview
As the starting point for folding, the properties of the denatured state ensemble are critical to the thermodynamics and kinetics of protein folding. We used methionine oxidation to destabilize monomeric λ repressor and predominantly populate the denatured state under non-denaturing buffer conditions. The denatured ensemble of lambda repressor comprises conformations that are compact. The hydrodynamic radius of the denatured state determined under physiological conditions by sedimentation velocity and pulsed-field gradient NMR is only slightly larger than the native state and much smaller than the highly-denatured state. These conformations are likely compact due to a significant degree of α-helical structure and some tertiary interactions indicated by circular dichroism. To determine which residues populate helical conformations, we used NMR spectroscopy. The chemical shift index (CSI) calculations for the Cα and Hα atoms indicate that the region of the protein corresponding to helix 1 in the native state has a significant α-helical population in the denatured state. Most of the short-range and medium-range assigned NOEs detected by NOESY spectroscopy are in this same region. There are NOEs in other regions of the protein, but they are not consistent with a highly helical conformations in any other region of the protein. No long-range NOEs were assigned. 15N NMR relaxation parameters were measured at two static magnetic fields to determine the backbone dynamics of the denatured state of monomeric lambda repressor. Reduced spectral density mapping and Lipari-Szabo model-free analysis were used to develop a molecular interpretation of the relaxation parameters. There are two regions of reduced conformational flexibility in this denatured state, with a region of flexibility between them. These two rigid regions correspond to protein sequence that consists of bulky amino acids that bury a large amount of surface area upon folding. Therefore, it is likely that steric hindrance leads to conformational restriction in unfolded proteins, not necessarily nascent secondary structure formation.
