Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy are analytical techniques employed for the analysis of protein secondary structure. The use of CD spectroscopy is limited to low protein concentrations (5 mg ml⁻¹). Here we introduce a quantum cascade laser (QCL)-based IR transmission setup for analysis of protein and polypeptide secondary structure at concentrations as low as 0.25 mg ml⁻¹in deuterated buffer solution. We present dynamic QCL-IR spectra of the temperature-induced α-helix to β-sheet transition of poly-L-lysine. The concentration dependence of the α-β transition temperature between 0.25 and 10 mg ml⁻¹was investigated by QCL-IR, FTIR and CD spectroscopy. By using QCL-IR spectroscopy it is possible to perform IR spectroscopic analysis in the same concentration range as CD spectroscopy, thus enabling a combined analysis of biomolecules secondary structure by CD and IR spectroscopy.

In this work, a novel EC-QCL-based setup for mid-IR transmission measurements in the amide I region is introduced for monitoring dynamic changes in secondary structure of proteins. For this purpose, α-chymotrypsin (aCT) acts as a model protein, which gradually forms intermolecular β-sheet aggregates after adopting a non-native α-helical structure induced by exposure to 50 % TFE. In order to showcase the versatility of the presented setup, the effects of varying pH values and protein concentration on the rate of β-aggregation were studied. The influence of the pH value on the initial reaction rate was studied in the range of pH 5.8–8.2. Results indicate an increased aggregation rate at elevated pH values. Furthermore, the widely accessible concentration range of the laser-based IR transmission setup was utilized to investigate β-aggregation across a concentration range of 5–60 mg mL⁻¹. For concentrations lower than 20 mg mL⁻¹, the aggregation rate appears to be independent of concentration. At higher values, the reaction rate increases linearly with protein concentration. Extended MCR-ALS was employed to obtain pure spectral and concentration profiles of the temporal transition between α-helices and intermolecular β-sheets. Comparison of the global solutions obtained by the modelled data with results acquired by the laser-based IR transmission setup at different conditions shows excellent agreement. This demonstrates the potential and versatility of the EC-QCL-based IR transmission setup to monitor dynamic changes of protein secondary structure in aqueous solution at varying conditions and across a wide concentration range. EC-QCL IR spectroscopy for monitoring protein conformation change