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

ALCARAZ M R; SCHWAIGHOFER A; GOICOECHEA H C; LENDL B

ANALYTICAL AND BIOANALYTICAL CHEMISTRY

SPRINGER HEIDELBERG

Lugar: HEIDELBERG; Año: 2016 vol. 408 p. 3933 - 3941

1618-2642

Abstract 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, -chymotrypsin (aCT) acts as a model protein, which gradually forms intermolecular -sheet aggregates after adopting non-native -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 -aggregation were studied. The influence of the pH value on the initial reaction rate was studied in the range of pH 5.8 - pH 8.2. Results indicate an increased aggregation rate at elevated pH values. Further, the wide 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 from 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 available concentration range.