Structural and Dynamical Properties of a Denatured Protein. Heteronuclear 3D NMR Experiments and Theoretical Simulations of Lysozyme in 8 M Urea
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- 作者：Harald Schwalbe ; Klaus M. Fiebig ; Matthias Buck ; Jonathan A. Jones ; Shaun B. Grimshaw ; Andrew Spencer ; Steffen J. Glaser ; Lorna J. Smith ; and Christopher M. Dobson
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：July 22, 1997
- 年：1997
- 卷：36
- 期：29
- 页码：8977 - 8991
- 全文大小：393K
- 年卷期：v.36,no.29(July 22, 1997)
- ISSN：1520-4995
文摘
Oxidized and reduced hen lysozyme denatured in 8 M ureaat low pH have been studied indetail by NMR methods. 15N correlated NOESY and TOCSYexperiments have provided near completesequential assignment for both 1H and 15Nresonances. Over 900 NOEs, including 130 (i,i + 2) and 23(i, i + 3) NOEs, could be identified byanalysis of the NOESY spectra of the denatured states, and3J(HN,H
) coupling constants and 15N relaxation rates have beenmeasured. The coupling constant and NOEdata were analyzed by comparisons with theoretical predictions from arandom coil polypeptide modelbased on amino acid specific 
,
distributions extracted from theprotein data bank. There is significantagreement between predicted and experimental NMR parameters suggestingthat local conformations ofthe denatured states are largely determined by short-range interactionswithin the polypeptide chain. Thisresult is supported by the observation that the chemical shift,coupling constant, and NOE data are littleaffected by whether or not the four disulfide bridge cross-links areformed in the denatured protein. Therelaxation data, however, show significant differences between theoxidized and reduced protein. Analysisof the relaxation data in terms of simple dynamics models providesevidence for weak clustering ofhydrophobic groups near tryptophan residues and increased barriers tomotion in the more compactconformers formed when the polypeptide chain is cross-linked by thedisulfide bridges. Using thisinformation, a structural description of these denatured states isgiven in terms of an ensemble of conformers,which have a complex relationship between their local and globalcharacteristics.
) coupling constants and 15N relaxation rates have beenmeasured. The coupling constant and NOEdata were analyzed by comparisons with theoretical predictions from arandom coil polypeptide modelbased on amino acid specific , distributions extracted from theprotein data bank. There is significantagreement between predicted and experimental NMR parameters suggestingthat local conformations ofthe denatured states are largely determined by short-range interactionswithin the polypeptide chain. Thisresult is supported by the observation that the chemical shift,coupling constant, and NOE data are littleaffected by whether or not the four disulfide bridge cross-links areformed in the denatured protein. Therelaxation data, however, show significant differences between theoxidized and reduced protein. Analysisof the relaxation data in terms of simple dynamics models providesevidence for weak clustering ofhydrophobic groups near tryptophan residues and increased barriers tomotion in the more compactconformers formed when the polypeptide chain is cross-linked by thedisulfide bridges. Using thisinformation, a structural description of these denatured states isgiven in terms of an ensemble of conformers,which have a complex relationship between their local and globalcharacteristics.