External Cavity\nQuantum Cascade Laser Vibrational\nCircular Dichroism Spectroscopy for Fast and Sensitive Analysis of\nProteins at Low Concentrations

Proteins are characterized by their complex levels of\nstructures,\nwhich in turn define their function. Understanding and evaluating\nthese structures is therefore crucial to illuminating biological processes.\nOne of the possible analytical methods is vibrational circular dichroism\n(VCD), which expands the structural sensitivity of classical infrared\n(IR) absorbance spectroscopy by the chiral sensitivity of circular\ndichroism. While this technique is powerful, it is plagued by low\nsignal intensities and long measurement times. Here we present an\noptical setup leveraging the high brilliance of a quantum cascade\nlaser to measure proteins in D<sub>2</sub>O at a path length of 204\nμm. It was compared to classical Fourier-transform infrared\nspectroscopy (FT-IR) in terms of noise levels and in its applicability\nto secondary structure elucidation of proteins. Protein concentrations\nas low as 2 mg/mL were accessible by the laser-based system at a measurement\ntime of 1 h. Further increase of the time resolution was possible\nby adapting the emission to cover only the amide I’ band. This\nallowed for the collection of spectral data at a measurement time\nof 5 min without a loss of performance. With this high time resolution,\nwe are confident that dynamic processes of protein can now be monitored\nby VCD, increasing our understanding of these reactions.