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M. Alcaraz, A. Schwaighofer, C. Kristament, G. Ramer, M. Brandstetter, H. Goicoechea, B. Lendl:
"EC-QC laser spectroscopy for IR transmission measurements of proteins in aqueous solution";
Poster: ICAVS 8, Wien; 12.07.2015 - 17.07.2015.

Infrared spectroscopy is a well-established experimental technique for the secondary structure analysis of proteins. The amide I band (1600-1700 cm-1) is most commonly used for analysis of the secondary structure, as differing patterns of hydrogen bonding, dipole-dipole interactions and geometric orientations in the α-helices, β-sheets, turns and random coil structures induce different frequencies of the C=O stretching vibrations in the peptide group that can be correlated with the respective secondary structural folding [1].

The most important difficulty of IR investigations of proteins in aqueous solution is the strong absorbance of the HOH-bending band that overlaps with the amide I band. Thus, for routinely used FTIR spectrometers employing low intensity thermal emitters as light source suitable path lengths for transmission measurements are restricted to 3-10 µm [3]. These low optical paths considerable impair the robustness of the system and do not allow experiments in flow through configuration.

We report IR transmission measurements of the protein amide I band in aqueous solution at large optical paths. A room-temperature operated tunable external-cavity quantum cascade laser (EC-QCL) allowed applying path length of up to 38 µm. An advanced data processing protocol was established to overcome fluctuations in the fine structure of the emission curve that are inherent to the employed EC-QCL due to its mechanical instabilities. To allow for wavenumber accuracy, a spectral calibration method has been elaborated to reference the acquired IR spectra to the absolute positions of the water vapor absorption bands.

mid-IR spectroscopy, external-cavity quantum cascade laser, protein structure.