A New Method to Determine the Transmembrane Conformation of Substrates in Intramembrane Proteolysis by Deep-UV Resonance Raman Spectroscopy.

We present a new method based on deep-UV resonance Raman spectroscopy to determine the backbone conformation of intramembrane protease substrates. The classical amide vibrational modes reporting on the conformation of just the transmembrane region of the substrate can be resolved from solvent exchangeable regions outside the detergent micelle by partial deuteration of the solvent. In the presence of isotopically triple-labeled intramembrane protease, these amide modes can be accurately measured to monitor the transmembrane conformation of the substrate during intramembrane proteolysis.

Mitigation of Rayleigh and Raman spectral interferences in multiway calibration of excitation-emission matrix fluorescence spectra.

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.