COMMUNICATION Raman Optical Activity of Filamentous Bacteriophages: Hydration of a-Helices Ewan W. Blanch 1 , Alasdair F. Bell 1 , Lutz Hecht 1 , Loren A. Day 2 * and Laurence D. Barron 1 * 1 Chemistry Department University of Glasgow Glasgow, G12 8QQ, UK 2 Public Health Research Institute, 455 First Avenue New York, NY, 10016, USA We report the ®rst observations of vibrational Raman optical activity (ROA) on intact viruses. Speci®cally, ROA spectra of the ®lamentous bac- teriophages Pf1, M13 and IKe in aqueous solution were measured in the range 600-1800 cm 1 . On account of its ability to probe directly the chiral elements of biomolecular structure, ROA has provided a new per- spective on the solution structures of these well-studied systems. The ROA spectra of all three are dominated by signatures of helical elements in the major coat proteins, as expected from pre-existing data. The helical elements generate strong sharp positive ROA bands at 1300 and 1342 cm 1 in H 2 O solution, but in 2 H 2 O solution the 1342 cm 1 bands disappear completely. The spectra are similar to those of polypeptides under conditions that produce a-helical conformations. Our present results, together with results from other studies, suggest that the positive 1342 cm 1 ROA bands are generated by a highly hydrated form of a-helix, and that the positive 1300 cm 1 bands originate in a-helix in a more hydrophobic environment. The presence of signi®cant amounts of highly hydrated helical sequences accords with the known ¯exibility of these viruses. Differences of spectral detail for Pf1, M13 and IKe demon- strate that ROA is sensitive to subtle variations of conformation and hydration within the major coat proteins, with M13 and IKe possibly con- taining more non-helical structure than Pf1. The ROA spectra of Pf1 at temperatures above and below that at which a structural transition is known to occur (10  C) reveal little difference in the protein confor- mation between the two forms, but there are indications of changes in DNA structure. # 1999 Academic Press Keywords: ®lamentous bacteriophages; Raman optical activity; coat protein conformation; a-helix hydration *Corresponding authors The ®lamentous bacteriophages are ¯exible rods approximately 800 to 2000 nm long and 6 to 7 nm in diameter, comprised of single-stranded circular DNA molecules surrounded by cylindrical protein shells. Each protein shell is a helical array of several thousand identical copies of a major coat protein subunit, of about 50 amino acid residues; the shell is terminated at both ends by a few copies of minor coat proteins. The packaged DNA genome constitutes from 6 to 14 % of the total vir- ion mass, depending on the viral strain. Obser- vations made with a variety of techniques, including X-ray and neutron diffraction from oriented ®bres as well as NMR, Raman, IR, ¯uor- escence and UV CD, have established that there are two types of fundamental helical symmetry for the protein shells, that the conformations of the individual major coat proteins are predominantly a-helical, that the positively charged C-terminal domain of each subunit interacts with the DNA, whereas its negatively charged N-terminal domain is on the virion exterior, and that there are striking differences in DNA conformation (for reviews, see Day et al., 1988a,b; Marvin, 1998). Since ®lamen- tous bacteriophages have not been crystallized, the E-mail addresses of the corresponding authors: laurence@chem.gla.ac.uk; day@phri.nyu.edu Abbreviations used: ROA, Raman optical activity; NMR, nuclear magnetic resonance spectroscopy; IR, infrared spectroscopy; UV CD, ultraviolet circular dichroism. Article No. jmbi.1999.2871 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 290, 1±7 0022-2836/99/260001±7 $30.00/0 # 1999 Academic Press