NMR quantum computing

Abstract:

The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH(2). The phi angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis-trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of(3)J(HNHalpha) values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of 'random coil' (1)H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that (13)C and(15)N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.

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.

Abstract:

Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with alpha-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.