Shining light on protein folding using mid-IR laser spectroscopy and chemometrics

Abstract

External cavity mid-IR quantum cascade lasers (EC-QCL) are coherent and polarized sources with fast intensity and wavelength modulation capabilities. They have revolutionized the way mid-IR spectrscopy of liquids, gases as well as solids (imaging) can be performed. Interaction of mid-IR laser radiation with analytes causes, in addition to attenuation of the light intensity, phase shifts due to anomaleous dispersion as well as heating of the sample as the absorbed photon energy is released into the sample matrix. This presentation will highlight the capability of mid-IR laser spectroscopy to provide direct, label-free information on protein secondary structures on a range of different examples. When coupled to liquid chromatography (ion exchange, size exclusion) protein separtions can be followed in-line and using MCR-ALS partially co-eluting proteins can be resolved [1,2]. Similary, MCR-ALS applied to data from protein melting experiments followed by EC-QCL spectroscopy provides structural information on intermediates [3]. Vibrational circular dichroism (VCD) expands the structural sensitivity of IR absorbance spectroscopy by the chiral sensitivity of infrared circular dichroism. While this technique is powerful, it is plagued by low signal intensities and long measurement times. Therefore, it is of utmost importance to select the most relevant wavelengths for measurement when time resolution is critical. Here, a setup for measurimg VCD spectra is introduced and results will be shown on the measurement of proteins in D2O [4]. Time-resolved monitoring of a racemisation process (R-,S-Binol) by VCD with high time resolution is achieved by application of Lasso [5]. Finally, AFM-IR will be introduced as a new mid-IR imaging technique achieving nanoscale spatial resolution. Applied to imaging of microorganism and using multivariate modelling the intracellular distribution of cell components can be revealed. This will be demonstrated by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside cells [6].