M. Alcaraz, A. Schwaighofer, H. Goicoechea, B. Lendl:
"EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of β-aggregation in alcohol-denaturated α-chymotrypsin";
Analytical and Bioanalytical Chemistry, 408 (2016), 15; S. 3933 - 3941.

Kurzfassung englisch:
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 intermol. 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 concn. 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 concn. range of the laser-based IR transmission setup was utilized to investigate β-aggregation across a concn. range of 5-60 mg mL-1. For concns. lower than 20 mg mL-1, the aggregation rate appears to be independent of concn. At higher values, the reaction rate increases linearly with protein concn. Extended MCR-ALS was employed to obtain pure spectral and concn. profiles of the temporal transition between α-helixes and intermol. β-sheets. Comparison of the global solns. obtained by the modeled 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 aq. soln. at varying conditions and across a wide concn. range.

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