EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of ?-aggregation in alcohol-denaturated ?-chymotrypsin.

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 a 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 5.8-8.2. 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 ?-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 ?-helices and intermolecular ?-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. Graphical abstract EC-QCL IR spectroscopy for monitoring protein conformation change.

SUBMITTER: Alcaraz MR 

PROVIDER: S-EPMC4873525 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature