Residual structure in unfolded proteins revealed by Raman optical activity

Wilson, G., Hecht, L. and Barron, L. D. (1996) Residual structure in unfolded proteins revealed by Raman optical activity. Biochemistry, 35(38), pp. 12518-12525. (doi:10.1021/bi961314v) (PMID:8823188)

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Because of its ability to probe directly the chiral elements of the peptide backbone, together with the very short time scale of the scattering process, vibrational Raman optical activity (ROA) can provide new information on structure in non-native states of proteins. Here we report ROA studies of hen egg white lysozyme and bovine ribonuclease A in unfolded denatured states, prepared by reducing all the disulfide bonds. ROA spectra of unfolded lysozyme at 45, 20, and 2 °C, and of unfolded ribonuclease A at 35 and 20 °C, are presented and discussed. At 45 and 20 °C, unfolded lysozyme appears to contain very little extended secondary structure, but at 2 °C there could be roughly 20% of the native amount of α-helix present but little β-sheet. Unfolded ribonuclease A, on the other hand, appears to contain roughly 50% of its native-like secondary structure, including both α-helix and β-sheet, at 20 °C; similar secondary structure persists at 35 °C, but the amount is reduced. The most striking result is the observation of three sharp ROA bands in the extended amide III region, originating in coupled Cα−H and N−H deformations, which might monitor directly the dominant intrinsic propensities for residues to adopt particular φ,ψ angles, averaged over the different amino acids in the mobile heteropolypeptide. Specifically, positive bands at ∼1300 and 1314 cm-1 appear to monitor propensities for α-helix and β-structure, respectively, and a negative band at ∼1237 cm-1 appears to monitor that for the poly(l-proline) II helix. These signals are generated by individual residues clustering in the most favorable regions of the Ramachandran plot and are present even in the absence of signals from the corresponding extended secondary structures. At 45 °C, the 1300 and 1314 cm-1 ROA bands of unfolded lysozyme coalesce into a single sharp band from which an analysis similar to that used for exchange effects in NMR suggests a rate of ∼2.6 × 1012 s-1 for interconversion between the individual residue conformations at this temperature.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Hecht, Dr Lutz and Barron, Professor Laurence
Authors: Wilson, G., Hecht, L., and Barron, L. D.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Biochemistry
Publisher:American Chemical Society
ISSN (Online):1520-4995

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