Laurence D. Barron: Raman Optical Activity
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pioneered the technique of Raman optical activity (ROA), which gives complete
vibrational optical activity spectra of chiral molecules via circularly
polarized Raman spectroscopy.
This subject started with the discovery during my doctoral work in Oxford with Peter Atkins of a new optical process involving interference between light waves scattered via the polarizability and optical activity tensors of a chiral molecule. This leads to a dependence of the scattered intensity on the degree of circular polarization of the incident beam and to a small circularly polarized component in the scattered beam. I subsequently developed the definitive theory of ROA with David Buckingham in Cambridge in 1971, and made the first observations (in the form of a small difference in the intensity of Raman scattering in right- and left-circularly polarized incident light) with David Buckingham and Martin Bogaard 1973.
The basic ROA experiment. A tiny difference in the intensity of Raman scattering from chiral molecules is measured in right and left circularly polarized incident laser light.
Breakthroughs in instrumentation achieved with Lutz Hecht in my Glasgow laboratory over the past decade have rendered biological molecules in aqueous solution accessible to ROA studies. Our current home-built instruments provide high quality ROA spectra of the entire range of chiral molecular species, from small chiral organics to intact viruses. ROA bears the same relationship to conventional Raman spectroscopy as does UVCD to conventional UV absorption spectroscopy and so is a new source of stereochemical information.
Comparison of experimental with ab initio theoretical ROA spectra is a new method for determining absolute configuration of small chiral molecules. A good example is our determination in 1997 of the absolute configuration CHFClBr, first prepared over 100 years ago but which had resisted assignment using standard methods. ROA also measures enantiomeric purity with an accuracy of ~0.1% and, unlike conventional methods, can be applied to individual components in mixtures of different chiral species.
A commercial ROA instrument is now available from BioTools, Inc. (www.btools.com). It is based on a new design due to Werner Hug (University of Fribourg, Switzerland) and is significantly more sensitive than our home-built Glasgow instruments. The availability of this instrument will facilitate the widespread application of ROA in chemistry and the life sciences.
Raman Optical Activity in Structural Biology
Because of its sensitivity to chirality, ROA is a powerful new probe of the aqueous solution structure and behaviour of biomolecules including peptides, proteins, carbohydrates, nucleic acids and viruses. For example, as well as peptide backbone bands arising from secondary structure such as a-helix and b-sheet, protein ROA spectra also contain bands from loops and turns and so can provide information about the three-dimensional solution structure. ROA is also able to distinguish hydrated and non-hydrated variants of a-helix and b-sheet. This plethora of structure-sensitive bands in protein ROA spectra (many more than in CD and FTIR), analyzed with the help of pattern recognition techniques, makes ROA valuable for rapid structure determination, including fold recognition, in areas such as structural genomics. Our results on non-native protein states, and on natively unfolded proteins associated with Parkinson's, Alzheimers and the prion diseases, demonstrate that ROA is also ideal for studying the 'conformational plasticity' associated with protein misfolding and disease. ROA can also recognize subtle differences in b-sheet structures that are important for understanding aberrant protein folding and for distinguishing different types of b-sheet folds in native proteins.
The Raman (top) and ROA (bottom) spectra of rabbit aldolase. The ROA band pattern reflects the a/b TIM barrel fold of this protein.
The power of ROA is strikingly demonstrated by our results on intact viruses. Most, including filamentous, helical and icosahedral types, are accessible to ROA studies, from which the folds of the major coat proteins can be deduced, together with the structure of the nucleic acid core in some cases. Details such as the absolute stereochemistry of tryptophan side chains, usually only available from high resolution X-ray crystallography, are also sometimes available. Although knowledge of virus structure at the molecular level is essential for understanding their modus operandi and hence for structure-guided antiviral drug design, etc., most of the thousands of different viruses are inaccessible to key structural biology techniques. X-ray structures at atomic resolution are of immense value but are only available for several dozen viruses: even then, only the coat protein structures are fully resolved, much of the nucleic acid being too disordered to be visible. ROA has already proved valuable in studies of potato virus X, which destroys large amounts of potato crops worldwide: almost nothing was known previously about its structure due to poor quality X-ray fibre diffraction data.
The Glasgow biomolecular ROA work was the subject of a recent feature article in Chemical and Engineering News (January 13, 2003, 36-39).
Rayleigh and Raman scattering from optically active molecules. L. D. Barron and A. D. Buckingham (1971). Mol. Phys. 20, 1111-1119.
Raman scattering of circularly polarized light by optically active molecules. L. D. Barron, M. P. Bogaard and A. D. Buckingham (1973). J. Am. Chem. Soc. 95, 603-605.
Determination of enantiomeric excess using vibrational Raman optical activity. L. Hecht, A. L. Phillips and L. D. Barron (1995). J. Raman Spectrosc. 26, 727-732.
Raman optical activity. L. Hecht and L. D. Barron (1996). In Modern Aspects of Raman Spectroscopy (ed. J. J. Laserna), Wiley, Chichester, pp 265-304.
Absolute configuration of bromochlorofluoromethane from experimental and ab initio theoretical vibrational Raman optical activity. J. Costante, L. Hecht, P. L. Polavarapu, A. Collet and L. D. Barron (1997). Ang. Chem. Int. Ed. Engl. 36, 885-887.
Solution structure and dynamics of biomolecules from Raman optical activity. L. D. Barron, L. Hecht, E. W. Blanch and A. F. Bell (2000). Prog. Biophys. Molec. Biol. 73, 1-49.
Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme. E. W. Blanch, L. A. Morozova-Roche, D. A. E. Cochran, A. J. Doig, L. Hecht and L. D. Barron (2000). J. Mol. Biol. 301, 553-563.
A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins. C. D. Syme, E. W. Blanch, C. Holt, R. Jakes, M. Goedert, L. Hecht and L. D. Barron. (2002). Eur. J. Biochem. 269, 148-156.
Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity. E. W. Blanch, D. J. Robinson, L. Hecht, C. D. Syme, K. Nielsen and L. D. Barron. J. Gen. Virol. 83, 241-246.
Molecular structures of viruses from Raman optical activity. E. W. Blanch, L. Hecht, C. D. Syme, V. Volpetti, G. P. Lomonossoff, K. Nielsen and L. D. Barron (2002). J. Gen. Virol. 83, 2593-2600.
Unfolded proteins studied by Raman optical activity. L. D. Barron, E. W. Blanch and L. Hecht (2002). Adv. Prot. Chem. 62, 51-90.
Structure and behaviour of proteins, nucleic acids and viruses from Raman optical activity. L. D. Barron, E. W. Blanch, I. H. McColl, C. D. Syme, L. Hecht and K. Nielsen (2003). Spectroscopy17, 101-126.
perspective on b-sheet structures using
vibrational Raman optical activity: From poly(L-lysine) to the prion protein. I.
H. McColl, E. W. Blanch, A. C. Gill, A. G. O. Rhie, M. A. Ritchie, L. Hecht, K.
Nielsen and L. D. Barron (2003). J. Am. Chem. Soc. 125,
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