STRUCTURAL AND DYNAMICAL PROPERTIES OF A DENATURED PROTEIN - HETERONUCLEAR 3D NMR EXPERIMENTS AND THEORETICAL SIMULATIONS OF LYSOZYME IN 8-M-UREA

Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. N-15 correlated NOESY and TOCS Y experiments have provided near complete sequential assignment for bo th H-1 and N-15 resonances. Over 900 NOEs, including 130 (i, i + 2) an d 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spe ctra of the denatured states, and (3)J(HN, Ha) coupling constants and N-15 relaxation rates have been measured. The coupling constant and NO E data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi d istributions extracted from the protein data bank. There is significan t agreement between predicted and experimental NMR parameters suggesti ng that local conformations of the denatured states are largely determ ined by short-range interactions within the polypeptide chain. This re sult 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 ox idized and reduced protein. Analysis of the relaxation data in terms o f simple dynamics models provides evidence for weak clustering of hydr ophobic groups near tryptophan residues and increased barriers to moti on in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a stru ctural description of these denatured states is given in terms of an e nsemble of conformers, which have a complex relationship between their local and global characteristics.