Deep-UV Resonance Raman Analysis of the Rhodobacter capsulatus Cytochrome bc1 Complex Reveals a Potential Marker for the Transmembrane Peptide Backbone
journal contributionposted on 2011-08-02, 00:00 authored by Christopher M. Halsey, Olayinka O. Oshokoya, Renee D. Jiji, Jason W. Cooley
Classical strategies for structure analysis of proteins interacting with a lipid phase typically correlate ensemble secondary structure content measurements with changes in the spectroscopic responses of localized aromatic residues or reporter molecules to map regional solvent environments. Deep-UV resonance Raman (DUVRR) spectroscopy probes the vibrational modes of the peptide backbone itself, is very sensitive to the ensemble secondary structures of a protein, and has been shown to be sensitive to the extent of solvent interaction with the peptide backbone [Wang, Y., Purrello, R., Georgiou, S., and Spiro, T. G. (1991) J. Am. Chem. Soc. 113, 6368−6377]. Here we show that a large detergent solubilized membrane protein, the Rhodobacter capsulatus cytochrome bc1 complex, has a distinct DUVRR spectrum versus that of an aqueous soluble protein with similar overall secondary structure content. Cross-section calculations of the amide vibrational modes indicate that the peptide backbone carbonyl stretching modes differ dramatically between these two proteins. Deuterium exchange experiments probing solvent accessibility confirm that the contribution of the backbone vibrational mode differences are derived from the lipid solubilized or transmembrane α-helical portion of the protein complex. These findings indicate that DUVRR is sensitive to both the hydration status of a protein’s peptide backbone, regardless of primary sequence, and its secondary structure content. Therefore, DUVRR may be capable of simultaneously measuring protein dynamics and relative water/lipid solvation of the protein.
DUVRRstructure contentDeuterium exchange experimentsstructure content measurementspeptide backbone carbonylTransmembrane Peptide BackboneClassical strategiespeptide backboneRhodobacter capsulatus cytochrome bc 1Rhodobacter capsulatus Cytochrome bc 1 Complexdetergent solubilized membrane proteinbackbone vibrational mode differencesamide vibrational modes