Abstract

Although optical activity in chiral molecules was discovered almost two centuries ago, it was not until the early 1970s that optical activity in vibrational spectra was observed. Thanks to progress in experiment, theory and computation, powerful chiroptical spectroscopies based on vibrational optical activity measurements have evolved rapidly. We review the basic theory and recent applications of the Raman and infrared approaches, emphasising the impact ab initio calculations are making on simulation of vibrational optical activity spectra to extract detailed structures, including the absolute configuration and conformer populations, of chiral molecular and biomolecular species, and discuss many new features that are emerging.

Abstract

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements present in unfolded states. Carbohydrate ROA spectra provide information on the central features of carbohydrate stereochemistry, especially that of the glycosidic link. Glycoprotein ROA spectra provide information on both the polypeptide and carbohydrate components. This article describes the ROA technique and presents and discusses the ROA spectra of a selection of proteins, carbohydrates, and a glycoprotein. The many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques, illustrated here using nonlinear mapping, to determine structural relationships between different proteins. © 2005 Wiley-Liss, Inc. Chirality 18:103–115, 2006.

Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine α-casein, soybean Bowman–Birk protease inhibitor, and rabbit Cd₇-metallothionein, all of which have irregular folds in the native state. The results show that ROA is able to distinguish between two types of disorder. Specifically, invertase, α-casein, the Bowman–Birk inhibitor, and metallothionein appear to possess a “static” type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more “dynamic” type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states. In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive ϕ,ψ angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran ϕ,ψ angles for each amino acid residue, giving rise to an ensemble of interconverting conformers. In both cases there is a propensity for the ϕ,ψ angles to correspond to the α, β and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds. Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix. Long sequences of relatively unconstrained PPII helix, as in α-casein, may impart a plastic (rheomorphic) character to the structure. © 2001 John Wiley & Sons, Inc. Biopoly 58: 138–151, 2001

Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine α-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22°C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at ∼1340 cm⁻¹, which is assigned to a hydrated form of α helix, is more similar to that in the ROA spectrum of bovine α-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine α-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at ∼1300 cm⁻¹ in hen lysozyme that is assigned to an α helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at ∼1305 cm⁻¹, which may reflect greater heterogeneity in some α-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22°C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22°C and the apo form at pH 4.5 and 48°C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated α helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine α-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at ∼1551 cm⁻¹ in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.