"Parallel factor analysis of multi-excitation ultraviolet resonance Raman spectra for protein secondary structure determination".

Protein secondary structural analysis is important for understanding the relationship between protein structure and function, or more importantly how changes in structure relate to loss of function. The structurally sensitive protein vibrational modes (amide I, II, III and S) in deep-ultraviolet resonance Raman (DUVRR) spectra resulting from the backbone C-O and N-H vibrations make DUVRR a potentially powerful tool for studying secondary structure changes. Experimental studies reveal that the position and intensity of the four amide modes in DUVRR spectra of proteins are largely correlated with the varying fractions of α-helix, ß-sheet and disordered structural content of proteins. Employing multivariate calibration methods and DUVRR spectra of globular proteins with varying structural compositions, the secondary structure of a protein with unknown structure can be predicted. A disadvantage of multivariate calibration methods is the requirement of known concentration or spectral profiles. Second-order curve resolution methods, such as parallel factor analysis (PARAFAC), do not have such a requirement due to the "second-order advantage." An exceptional feature of DUVRR spectroscopy is that DUVRR spectra are linearly dependent on both excitation wavelength and secondary structure composition. Thus, higher order data can be created by combining protein DUVRR spectra of several proteins collected at multiple excitation wavelengths to give multi-excitation ultraviolet resonance Raman data (ME-UVRR). PARAFAC has been used to analyze ME-UVRR data of nine proteins to resolve the pure spectral, excitation and compositional profiles. A three factor model with non-negativity constraints produced three unique factors that were correlated with the relative abundance of helical, ß-sheet and poly-proline II dihedral angles. This is the first empirical evidence that the typically resolved "disordered" spectrum represents the better defined poly-proline II type structure.

Role of bilayer characteristics on the structural fate of aß(1-40) and aß(25-40).

The ß-amyloid (Aß) 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 Aß is needed. Employing a combination of circular dichroism and deep-UV resonance Raman spectroscopies, Aß(25-40) was found to fold spontaneously upon association with anionic lipid bilayers. The hydrophobic portion of the disease-related Aß(1-40) peptide, Aß(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 Aß(25-40) peptide was found to depend on both the hydrophobic thickness of the bilayer and the duration of incubation. Similarly, the disease-related Aß(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 ß-sheet structures over longer time frames. ß-Sheet structure is formed prior to helical unwinding, implying a model in which ß-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 Aß(1-40) from the membrane surfaces following its formation by cleavage of APP within the membrane.

Deep-UV resonance Raman analysis of the Rhodobacter capsulatus cytochrome bc1complex reveals a potential marker for the transmembrane peptide backbone.

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.