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 abstractEC-QCL IR spectroscopy for monitoring protein conformation changeElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9464-5) contains supplementary material, which is available to authorized users.

pH titration of β-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis

“…The high available emission powers enabled to increase the optical path up to 38 µm for transmission measurements in the protein region [12]. EC-QCL-based IR transmission measurements have been successfully accomplished for the analysis of protein secondary structure [13][14][15]. Furthermore, the feasibility of protein discrimination and quantitation in commercial bovine milk samples has been demonstrated by QCL-IR spectroscopy and evaluation of the amide I band using partial least squares (PLS) modelling [16][17][18][19].…”

“…Due to its flexibility and robustness, it has been successfully used in combination with various analytical techniques, such as chromatography [41], electrophoresis [42], and flow analysis [43], among others. In combination with spectroscopic techniques, particularly with IR spectroscopy, MCR-ALS has become a highly valuable technique for the study of evolving processes [13,44,45].…”

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations

“…Application of these high-emission power light sources has allowed to significantly increase the optical path for transmission measurements 23 . Most recently, EC-QCL-based IR transmission measurements have been accomplished for the analysis of protein secondary structure in aqueous solution 24 25 .…”

Recent advancements of EC-QCL based mid-IR transmission spectroscopy of proteins and application to analysis of bovine milk1

“…This arising spectral pattern is commonly attributed to intermolecular antiparallel βsheet aggregates, frequently occurring in thermally denatured proteins [22,41]. The effect of different pH values and protein concentration on the kinetics of β-sheets aggregation has been investigated employing QCL-IR spectroscopy [2].…”