VIRUS STRUCTURE BY LASER RAMAN SPECTROSCOPY .45. RAMAN SPECTROSCOPY OF THE FILAMENTOUS VIRUS FF (FD, FL, M13) - STRUCTURAL INTERPRETATION FOR COAT PROTEIN AROMATICS

摘要

Site-specific isotope substitutions in the coat protein (pVIII) of the filamentous bacterial virus Ff (fd, f1, M13) have been employed to advance vibrational band assignments and facilitate structural interpretation of the Raman spectrum. We report spectra of phage fd assembled in vivo from pVIII subunits incorporating either deuteriophenylalanine (F-d5), deuteriotryptophan (W-d5), or deuteriotyrosine (Y-d4) residues with labeled ring sites. The deuterated aromatics were introduced into fd individually and in combination. On the basis of observed isotope shifts, definitive assignments have been developed for all prominent Raman bands diagnostic of the pVIII aromatic residues (F11, F42, F45, W26, Y21, Y24). The present study constitutes the first direct experimental determination of Raman fingerprints of tyrosine and phenylalanine side chains within hydrophobic alpha-helical domains and yields unexpected results. Importantly, neither Y21 nor Y24 of pVIII exhibits the ''canonical'' Fermi doublet expected in the 820-860 cm(-1) interval of the Raman spectrum. Instead, each tyrosine exhibits a single band near 853 cm(-1). Since the application of denaturing conditions is sufficient to generate in fd an apparent Fermi doublet, it is concluded that the anomalous singlet is intrinsic to tyrosine environments in the native virion assembly. In addition, the Raman results clearly demonstrate an interdependence of the environments of aromatic side chains in virion subunits. We show that the results on fd isotopomers are also confirmed by Raman spectroscopy of Ff virions incorporating the tyrosine mutations Y21M, Y24M, and Y21F/Y24S. The Raman marker bands identified for pVIII aromatics modify and extend Raman correlations proposed previously for proteins. The unusual environments detected for aromatic residues in the mature Ff assembly are discussed in relation to recently proposed models for filamentous virion architecture.