Raman markers of nonaromatic side chains in an alpha-helix assembly: Ala, Asp, Glu, Gly, Ile, Leu, Lys, Ser, and Val residues of phage fd subunits

The study of filamentous virus structure by Raman spectroscopy requires acc urate band assignments. In previous work, site- and residue-specific isotop e substitutions were implemented to elucidate definitive assignments for Ra man bands arising from vibrational modes of the alpha-helical coat protein main chain and aromatic side chains in the class I filamentous phage, fd [O verman, S. A., and Thomas, G. J., Jr. (1995) Biochemistry 34, 5440-5451; Ov erman, S. A., and Thomas, G. J., Jr. (1998) Biochemistry 37, 5654-5665]. He re, we extend the previous methods and expand the assignment scheme to iden tify Raman markers of nonaromatic side chains of the coat protein in the na tive fd assembly. This has been accomplished by Raman analysis of Il differ ent fd isotopomers selectively incorporating deuterium at specific sites in either alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine , lysine, serine, or valine residues of the coat protein. Raman markers are also identified for the corresponding deuterated side chains. In combinati on with previous assignments, the results provide a comprehensive understan ding of coat protein contributions to the Raman signature of the fd virion and validate Raman markers assigned to the packaged single-stranded DNA gen ome. The findings described here show that nonaromatic side chains contribu te prolifically to the fd Raman signature, that marker bands for specific n onaromatics differ in general from those observed in corresponding polypept ides and amino acids, and that the frequencies and intensities of many nona romatic markers are sensitive to secondary and higher-order structures. Non aromatic markers within the 1200-1400 cm(-1) interval also interfere seriou sly with the diagnostic Raman amide III band that is normally exploited in secondary structure analysis. Implications of these findings for the assess ment of protein conformation by Raman spectroscopy are considered.