Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperaturejump (T-jump) was used to investigate early steps in the temperature induced α-helix to β-sheet conformational transition of poly(L-lysine) (poly(K)). Excitation at 197 nm from a tunable frequency-quadrupled Ti:Sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although unionized poly(K) (pH 11.6) is mainly α-helical below 30°C, there is a detectable fraction (∼15 %) of unfolded polypeptide, which is mainly in the polyproline II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be β-strand. Above 30°C unionized poly(K) forms β-sheet at a rate (minutes) which increases with increasing temperature. A 22 -44°C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/β-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, ∼ 2 % at the 3 μs time limit of the experiment. The rise time of the PPII signals is ∼250 ns, consistent with melting of short α-helical segments. A model is proposed in which the melted PPII segments interconvert with β-strand conformation, whose association through inter-strand H-bonding nucleates the formation of β-sheet. The intrinsic propensity for β-strand formation could be a determinant of β-sheet induction time, with implications for the onset of amyloid diseases. KeywordsUVRR; resonance Raman; protein folding; poly(L-lysine); PPII; β-strand; α-helix; β-sheet; temperature-jump; T-jump Several degenerative diseases, including Alzheimer's, Huntington's, Parkinson's, and transmissible spongiform encephalopathies (TSE), are associated with misfolding and aggregation of proteins (1). The misfolded proteins form fibrillous aggregates, termed amyloid fibrils, which have a cross β-sheet structure in which the polypeptide backbone is perpendicular to the fibril axis(2). This allows the fibril to grow lengthwise with the addition of subsequent misfolded protein molecules. It is interesting to note that conditions which promote partial folding of proteins, appear to promote fibril formation in vitro (3)(4)(5). † This work was supported by NIH Grants R01 GM 025158 to T.G.S. and NIH F32 AG 022770 to R.D.J. * To whom correspondence should be addressed: Princeton University, Department of Chemistry, Princeton, NJ, 08544. Telephone: (609) Certain proteins with abundant α-helical structure, including the prion protein (PrP C ) and apomyoglobin, convert to β-sheet rich structures under denaturing conditions (4-6). What is equally provocative is the correlation between metastable α-helices and β-sheet amyloid formation. The β-amyloid (Aβ) peptide involved in Alzheimer's disease and its constituent fragments contain no stable secondary structure in aqueous solutions (7,8), but when the nascent helic...

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A weighted parallel factor analysis (W-PARAFAC) model is applied to excitation-emission matrix (EEM) fluorescence spectra of carbamate pesticides to aid with calibration in the presence of Raman scattering. Traditional PARAFAC inefficiently models the Raman scattering, resulting in prediction and calibration errors when a significant background is present. Four different weighting strategies were investigated and compared with subtraction of the appropriate sample background. Using a binary weighting strategy produced superior results, compared with a continuous distribution of weights. Further choice of weighting strategies, which are optimized to include either maximum analyte signal or to exclude a maximum amount of background scattering, is dependent on the degree of overlap and relative signal intensity.

We compare the freezing/melting behavior of water hydrating single-supported bilayers of a zwitterionic lipid DMPC with that of an anionic lipid DMPG. For both membranes, the temperature dependence of the elastically scattered neutron intensity indicates distinct water types undergoing translational diffusion: bulk-like water probably located above the membrane and two types of confined water closer to the lipid head groups. The membranes differ in the greater width ΔT of the water freezing transition near the anionic DMPG bilayer (ΔT ∼ 70 K) compared to zwitterionic DMPC (ΔT ∼ 20 K) as well as in the abruptness of the freezing/melting transitions of the bulk-like water.

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.