title: 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.
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, 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. Graphical abstract EC-QCL IR spectroscopy for monitoring protein conformation change.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060, Vienna, Austria.|Laboratorio de Desarrollo Analítico y Quimiometría, FBCB, Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, 3000, Santa Fe, Argentina.
authors.forename: Mirta R
authors.initials: MR
authors.lastname: Alcaráz
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwaighofer
authors.affiliation: Laboratorio de Desarrollo Analítico y Quimiometría, FBCB, Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, 3000, Santa Fe, Argentina.
authors.forename: Héctor
authors.initials: H
authors.lastname: Goicoechea
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060, Vienna, Austria. bernhard.lendl@tuwien.ac.at.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Lendl
mesh_terms: D002918:Chymotrypsin / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D054023:Lasers, Semiconductor; D066329:Protein Aggregates; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods*
chemical_list: D066329:Protein Aggregates; D000431:Ethanol; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: 2,2,2-Trifluoroethanol; Aggregation; Infrared spectroscopy; Multivariate curve resolution-alternating least squares; Protein secondary structure; Quantum cascade laser