SOLUTION STRUCTURE AND DYNAMICS OF PEPTIDES AND PROTEINS FROM RAMAN OPTICAL ACTIVITY

V. Baumruk,¹ J. Kapitán,^1,2 P. Bouř²

¹ Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 121 16 Prague 2, Czech Republic, e-mail: baumruk@karlov.mff.cuni.cz

² Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic

In the postgenomic era, an increasing demand for structural information about biomolecules has appeared. This is partially met by the high-resolution nuclear magnetic resonance (NMR) spectroscopy and the X-ray crystallography. They are currently invaluable and will continue to be appreciated in the field of structural biology due to their ability to reveal structural details at the atomic resolution. However, there are limitations of their applicability. For example, many proteins are difficult to crystallize, and many others are too large or complicated to be currently approached by NMR. Although vibrational spectroscopic techniques do not yield the three-dimensional structures of proteins resolved at the atomic resolution directly, they provide useful alternative or supplementary information of these systems. Local arrangements of functionally important groups within macromolecules, or changes in these local structures that may relate to biological functions, are typical examples that can be conveniently probed by the vibrational spectroscopy.

A particularly informative method of vibrational spectroscopy is Raman optical activity (ROA) which refers to a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light [1]. ROA spectrum of a chiral molecule can contain up to 3N-6 fundamental bands (N is the number of atoms), each associated with one of the normal modes of vibration. These contain information about conformation and absolute configuration of the particular part of the structure embraced by the normal mode.

The data obtained by Raman spectroscopy and ROA are complementary, as both Raman and ROA scattering are generated by related, yet subtly different, mechanisms. For proteins the ROA spectra tend to be dominated by the bands arising mainly from polypeptide backbone, detailing thus the secondary and tertiary structure. On the other hand, Raman spectra, which are insensitive to chirality, tend to be dominated by bands from sidechains that are particularly sensitive to local environment. However, the principal problem with analysis of both Raman and ROA spectra is in obtaining definitive assignments for all component bands in a spectrum, because of band overlaps and a lack of satisfactory models for some structural motifs. Information obtained by both the techniques contains qualitatively different information than that yielded by NMR spectroscopy, because the time scale of Raman scattering event is much shorter than that of the fastest conformational fluctuation in biomolecules. The vibrational spectrum represents a simple superposition (weighted sum) of the spectra of all the conformers present in the sample.

It has been shown that ROA is an excellent technique for studying the polypeptide and protein structures in aqueous solution [2]. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide backbone, hydration, side-chain conformation, and structural elements present in denatured states. Backbone vibrations in polypeptides and proteins are usually associated with three main regions of the Raman spectrum: (i) the backbone skeletal stretch region (~870-1150 cm^-1); (ii) the extended amide III region (~1230-1340 cm^-1); and (iii) the amide I region (~1630-1700 cm^-1) [3]. The extended amide III region is particularly important for ROA studies because the coupling between N-H and C_α-H deformations is very sensitive to geometry and generates rich ROA band structure [4]. There are clear differences between ROA spectra of α-helix, β-sheet and disordered conformations of model polypeptides that enable ROA to discriminate between ordered and disordered polypeptide sequences in folded proteins [5].

In addition to ROA bands characteristic for α-helix and β-sheet, sharp ROA bands have been observed in the extended amide III that appear to originate from loops and turns. It can be explained as a consequence of the fact that ROA reflects the local geometry of individual residues due to the local short-range nature of the vibrational coupling [5]. Because of its ability to detect loop and turn structure, ROA is able to monitor changes in the protein fold that are inaccessible to conventional techniques [6] and give interesting results even on the partially unfolded states associated with reduced proteins and molten globules [7]. Unfolded proteins which are often difficult to investigate by means of high-resolution techniques have proven an especially fruitful object of investigation for ROA spectroscopy as many of the proteins important for proteomics are expected to be relatively unstructured [2].

Analyses of the structural information content from ROA spectra of biological systems have ranged from totally empirical correlation to fully theoretical predictions. For proteins, PCA (principal component analysis) based pattern recognition approach to identify protein folds from ROA spectral band shapes is under development [8] while at least for short peptides, the full ab initio quantum mechanical ROA simulations are possible [9].

The aim of this work is not only to give an overview of fruitful application of ROA in peptide and protein science but also present very recent results from our laboratory.

Ministry of Education of the Czech Republic is gratefully acknowledged for support (No. MSM 0021620835).

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