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.

No abstract

No abstract

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display the entire known spectral and redox variations known in the type 1 copper site. C. aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon.

No abstract

No abstract

Despite the various means available that can determine protein structure inside a lipid bilayer, there is still a need for rapid, inexpensive, and information-rich technique. Deep-UV resonance Raman (DUVRR) spectroscopy addresses this need and does not require laborious sample preparation. DUVRR spectroscopy is a mature technique that has primarily been applied for studies on water-soluble proteins. Early progress in characterizing the secondary structure of one membrane protein, cytochrome bc1, led to exploring other membrane proteins. DUVRR is not limited to membrane proteins, but also protein-like structures such as the potassium carrier valinomycin. We find DUVRR spectroscopy characterizes membrane protein structure as well as water-soluble protein structure. Additionally, it can describe the degree to which the protein is embedded into the lipid bilayer. These findings are promising for scientists wanting to understand the shape of proteins in the lipid membrane. There is also a need for further investigation of the variety of secondary structures.

is temperature and almost quantitative refolding of the KvAP channel is observed at 80 o C. In order to differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the KvAP channel provides a good model system to investigate the folding of a multi-domain integral membrane protein.