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Raman spectroscopy of the filamentous virus Ff (fd, fl, M13): structural interpretation for coat protein aromatics
1Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Abstract:
Site-specific isotope substitutions in the coat protein (pVIII) of the filamentous bacterial virus Ff (fd, fl, 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 (Fd5), deuteriotryptophan (Wd5), or deuteriotyrosine (Yd4) 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.
Insights
This study uses isotope substitutions in filamentous phage fd coat protein to assign Raman spectral bands. Unexpected tyrosine signals reveal unique environments within the native viral structure.
Area of Science:
- Biophysics
- Structural Biology
- Spectroscopy
Background:
- Filamentous phage coat protein (pVIII) structure and function are crucial for viral assembly.
- Raman spectroscopy is a powerful tool for analyzing molecular vibrations and protein structure.
- Understanding aromatic residue environments provides insights into protein folding and interactions.
Purpose of the Study:
- To assign specific Raman spectral bands to aromatic residues in the Ff phage coat protein (pVIII).
- To investigate the structural environment of tyrosine and phenylalanine residues in the native viral assembly.
- To establish Raman spectroscopic markers for aromatic side chains in filamentous virions.
Main Methods:
- Site-specific isotope substitutions (deuteration) of phenylalanine, tryptophan, and tyrosine residues in the Ff phage coat protein (pVIII).
- Raman spectroscopy of assembled Ff phage particles with isotopically labeled subunits.
- Analysis of isotope shifts to determine band assignments.
- Comparison with Raman spectra of Ff virions with specific tyrosine mutations.
Main Results:
- Definitive assignments were made for prominent Raman bands associated with pVIII aromatic residues (F11, F42, F45, W26, Y21, Y24).
- Tyrosine residues (Y21, Y24) in the native Ff virion exhibited a single Raman band near 853 cm-1, not the expected Fermi doublet.
- This anomalous singlet is intrinsic to the native tyrosine environment and distinct from denatured states.
- Raman results demonstrated interdependence between aromatic side chain environments within virion subunits.
Conclusions:
- The study provides the first direct experimental determination of Raman fingerprints for tyrosine and phenylalanine side chains in hydrophobic alpha-helical domains of Ff phage.
- The unusual tyrosine signal indicates a unique, constrained environment within the native Ff virion assembly.
- Identified Raman marker bands extend existing correlations for proteins and offer new insights into filamentous virion architecture.