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, MIRTA R.; SCHWAIGHOFER, ANDREAS; GOICOECHEA, HÉCTOR; LENDL, BERNHARD

ANALYTICAL AND BIOANALYTICAL CHEMISTRY

SPRINGER HEIDELBERG

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

1618-2642

A novel EC-QCL based setup for mid-IR transmission measurements in the amide I region was applied to monitor dynamic changes in secondary structure of the model protein -chymotrypsin (aCT). After adopting a non-native -helical structure induced by exposure to 50 % TFE, aCT gradually forms intermolecular -sheet aggregates. The influence of the pH value on the initial reaction rate was studied in the physiological relevant pH range (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. The results obtained by MCR-ALS compare well with evaluation of experimental IR spectra. This demonstrates the potential 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.