Deep-UV Resonance Raman Analysis of the Rhodobacter capsulatus Cytochrome bc₁ Complex Reveals a Potential Marker for the Transmembrane Peptide Backbone

Abstract

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 bc(1) 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.

Label‐Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches

“…Aromatic amino acids are precursors for various biologically important molecules,s uch as neurotransmitters and hormones.B ys electively investigating these biomolecules,i t would be possible to directly address certain metabolic pathways.F urthermore,i tcan be postulated that the overall composition of these molecular species can be used to identify tissue conditions or cellular states.M ajor advantages of UV resonance Raman spectroscopy are also the absence of af luorescence background and the signal enhancement by the resonance effect, which improve the signal quality and lead to fast data acquisition times.E arly attempts to inves-tigate biological samples were reported in the late 1990s, when the first UVRR data of viruses were reported, along with assignments of the observed Raman bands at different UV excitation wavelengths. [157] Multiwavelength UVRR was employed to spectroscopically characterize different growth phases of bacteria. [155] Nucleotide distributions were observed with resonant bands attributable to guanine and adenine.I n2 004, Jarvis and Goodacre demonstrated the feasibility of UVRR for the discrimination of bacteria of the urinary tract.…”

“…Deep-UVRR was used to study the interactions of the Rhodobacter capsulatus cytochrome bc1 complex, at ransmembrane protein that is sensitive to conformational changes. [157] Multiwavelength UVRR was employed to spectroscopically characterize different growth phases of bacteria. [158] However, compared to resonance Raman spectroscopy in the visible region, reports on cell and tissue samples are less frequent, probably owing to the susceptibility of biological samples to UV radiation.…”

Insights into Protein Structure and Dynamics by Ultraviolet and Visible Resonance Raman Spectroscopy

“…34 A small number of other detergent-solubilized membrane proteins have also been investigated, such as photosystem II, 138 cytochrome c oxidase, 139 and cytochrome bc 1 complex. 140 In the past several years, UVRR experiments have been extended to a membrane protein embedded in a synthetic lipid bilayer. 141,142 In particular, UVRR studies of outer membrane protein A (OmpA) and its mutants revealed tryptophan–lipid interactions during the insertion and folding of OmpA into bilayers of small unilamellar vesicles.…”

Proton-Coupled Electron Transfer and Redox-Active Tyrosines: Structure and Function of the Tyrosyl Radicals in Ribonucleotide Reductase and Photosystem II

“…54 Through resonance enhancement, UV probe wavelengths select for contributions from aromatic species in proteins. 55,56 These species include tyrosine and tryptophan and their corresponding radicals {for recent examples, see 57–61 }. To avoid UV damage, a flowing sample is employed.…”