Structural and Dynamical Properties of a Denatured Protein. Heteronuclear 3D NMR Experiments and Theoretical Simulations of Lysozyme in 8 M Urea

Abstract

Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. 15N correlated NOESY and TOCSY experiments have provided near complete sequential assignment for both 1H and 15N resonances. Over 900 NOEs, including 130 (i, i + 2) and 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spectra of the denatured states, and 3J(HN, Halpha) coupling constants and 15N relaxation rates have been measured. The coupling constant and NOE data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi distributions extracted from the protein data bank. There is significant agreement between predicted and experimental NMR parameters suggesting that local conformations of the denatured states are largely determined by short-range interactions within the polypeptide chain. This result is supported by the observation that the chemical shift, coupling constant, and NOE data are little affected by whether or not the four disulfide bridge cross-links are formed in the denatured protein. The relaxation data, however, show significant differences between the oxidized and reduced protein. Analysis of the relaxation data in terms of simple dynamics models provides evidence for weak clustering of hydrophobic groups near tryptophan residues and increased barriers to motion in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a structural description of these denatured states is given in terms of an ensemble of conformers, which have a complex relationship between their local and global characteristics.

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from antarctic cod

“…It is not possible therefore to provide a picture of a unique conformation for this peptide in solution, nor is it meaningful to do so. A complete description of the glycopeptide would have to take into account in detail the conformational averaging, and would involve an ensemble approach such as recently described for denatured lysozyme (Schwalbe et al, 1997). However, because the ATPA subsequences do have a preferred conformation, it is useful to focus on them, as it is possible that the disaccharide moiety interacts with the ice crystal or membrane surface.…”

“…The NOESY spectrum shows that the NH of the acetamide side chain of the GalNac residues has a large coupling constant (3J > (Wuthrich, 1986;Schwalbe et al, 1997). However, the rate of exchange of the Ala NH with water, as observed in the ROESY spectrum, is for most residues considerably greater than for the threonine residues, or the NHAc in the GalNAc residues.…”

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

“…In both its nonphosphorylated and phosphorylated states, the R region has R 2 relaxation rates generally in the range of 4-8 s -1 , consistent with the values expected for a disordered protein and lower than those expected for a folded protein with the same number of residues. Uniformly low R 2 relaxation rates throughout the sequence, with further reduced rates at the termini, are expected for a model disordered protein with no structural contacts 29 . In contrast, the R 2 relaxation rates for the R region are not uniform throughout the sequence: higher rates were observed for a number of residues, including His667, Glu733, Ser768 and Ser809, that are within regions having >5% helical population and close to phosphorylation sites.…”

“…In contrast, the R 2 relaxation rates for the R region are not uniform throughout the sequence: higher rates were observed for a number of residues, including His667, Glu733, Ser768 and Ser809, that are within regions having >5% helical population and close to phosphorylation sites. This suggests that the restriction of motion by secondary structural contacts contributes to higher R 2 relaxation rates, with potential additional contributions from the exchange of individual conformers between different conformational states, tertiary contacts or hydrophobic clusters 29 . Phosphorylation generally decreases R 2 relaxation rates (see difference between …”

Long-Range Correlated Dynamics in Intrinsically Disordered Proteins

“…29,31,32 Surprisingly, measurements performed on the IDP α-synuclein also showed a small dispersion at ωτ R = 1 with a correlation time comparable to the reorientation time calculated for a folded protein with the same molecular weight. 29 The dispersion should not be observed if α-synuclein was only subject to segmental motions with correlation times much shorter than the reorientation time of the protein, 16,33 since the dispersions corresponding to such times occur at much larger fields. The observed dispersion therefore suggested that a residual correlated motion is present in α-synuclein.…”