Residual Structure in Unfolded Proteins Revealed by Raman Optical Activity

Abstract

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 degrees C, and of unfolded ribonuclease A at 35 and 20 degrees C, are presented and discussed. At 45 and 20 degrees C, unfolded lysozyme appears to contain very little extended secondary structure, but at 2 degrees C there could be roughly 20% of the native amount of alpha-helix present but little beta-sheet. Unfolded ribonuclease A, on the other hand, appears to contain roughly 50% of its native-like secondary structure, including both alpha-helix and beta-sheet, at 20 degrees C; similar secondary structure persists at 35 degrees 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 C alpha-H and N-H deformations, which might monitor directly the dominant intrinsic propensities for residues to adopt particular phi, psi angles, averaged over the different amino acids in the mobile heteropolypeptide. Specifically, positive bands at approximately 1300 and 1314 cm-1 appear to monitor propensities for alpha-helix and beta-structure, respectively, and a negative band at approximately 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 degrees 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 approximately 2.6 x 10(12) s-1 for interconversion between the individual residue conformations at this temperature.

Helix, Sheet, and Polyproline II Frequencies and Strong Nearest Neighbor Effects in a Restricted Coil Library

“…Beyond PPII being the dominant backbone conformation in peptides (4,(26)(27)(28)(29)(30)(31)(32)(33), Adzhubei and Sternberg found the presence of an average of over one stretch of four consecutive PPII residues in a library of 80 proteins (42). The origin of the preference for PPII in peptides may be due to preferential solvation (36)(37)(38), a suggestion that is consistent with the stretches of PPII tending to occur on the surfaces of these proteins.…”

Peptide‐plane flipping in proteins

“…In reality, in the unfolded protein, peptide-plane atoms will form hydrogen bonds to water molecules and therefore not only will the energetics be influenced by the presence of water, but the kinetics of peptide-plane flipping will be intimately connected to the kinetics of water. Although we have no direct information on the rate constants for peptide-plane flips, the Raman Optical Activity work of Wilson et al (1996) on proteins in non-native states indicates that individual residues flicker between ␣, ␤, and poly(L-proline) II regions at a rate of ∼10 12 sec −1 . This paper presents the results of a study of peptide-plane flips using native state protein structures.…”

Host−Guest Study of Left-Handed Polyproline II Helix Formation

“…As many as 10% of all residues are found in the PPII conformation, although not necessarily as part of PPII helices (10). PPII helices have also been hypothesized to be a major component of protein denatured states (11)(12)(13)(14), giving them a role in a most fundamental process. Recently, Blanch et al (15) have suggested that the PPII helix might be the precursor conformation in amyloid formation.…”