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

Determination of protein secondary structure (α-helical, β-sheet, and disordered motifs) has become an area of great importance in biochemistry and biophysics as protein secondary structure is directly related to protein function and protein related diseases.

The application of UV excitation sources coupled with resonance Raman have the potential to offer information unavailable with the current inventory of commonly used structural techniques including X-ray, NMR and IR analysis. However, for ultraviolet resonance Raman (UVRR) spectroscopy to become a mainstream method for the determination of protein secondary structure content and monitoring protein dynamics, the application of multivariate data analysis methodologies must be made routine. Typically, the application of higher order data analysis methods requires robust pre-processing methods in order to standardize the data arrays. The application of such methods can be problematic in UVRR datasets due to spectral shifts arising from day-to-day fluctuations in the instrument response. Additionally, the non-linear increases in spectral resolution in wavenumbers (increasing spectral data points for the same spectral region) that results from increasing excitation wavelengths can make the alignment of multi-excitation datasets problematic. Last, a uniform and standardized methodology for the subtraction of the water band has also been a systematic issue for multivariate data analysis as the water band overlaps the amide I mode. Here we present a two-pronged preprocessing approach using correlation optimized warping (COW) to alleviate spectra-to-spectra and day-to-day alignment errors coupled with a method whereby the relative intensity of the water band is determined through a least-squares determination of the signal intensity between 1750 and 1900 cm(-1) to make complex multi-excitation datasets more homogeneous and usable with multivariate analysis methods.

The β-amyloid (Aβ) peptide is derived from the transmembrane (TM) helix of the amyloid precursor protein (APP) and has been shown to interact with membrane surfaces. To understand better the role of peptide-membrane interactions in cell death and ultimately in Alzheimer's disease, a better understanding of how membrane characteristics affect the binding, solvation, and secondary structure of Aβ is needed. Employing a combination of circular dichroism and deep-UV resonance Raman spectroscopies, Aβ(25-40) was found to fold spontaneously upon association with anionic lipid bilayers. The hydrophobic portion of the disease-related Aβ(1-40) peptide, Aβ(25-40), has often been used as a model for how its legacy TM region may behave structurally in aqueous solvents and during membrane encounters. The structure of the membrane-associated Aβ(25-40) peptide was found to depend on both the hydrophobic thickness of the bilayer and the duration of incubation. Similarly, the disease-related Aβ(1-40) peptide also spontaneously associates with anionic liposomes, where it initially adopts mixtures of disordered and helical structures. The partially disordered helical structures then convert to β-sheet structures over longer time frames. β-Sheet structure is formed prior to helical unwinding, implying a model in which β-sheet structure, formed initially from disordered regions, prompts the unwinding and destabilization of membrane-stabilized helical structure. A model is proposed to describe the mechanism of escape of Aβ(1-40) from the membrane surfaces following its formation by cleavage of APP within the membrane.