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.
Highlights
Infrared spectroscopy is a powerful and established analytical method to study the structure of proteins [1]
An EC-quantum cascade lasers (QCL)-based IR transmission setup has been introduced for application in secondary structure analysis of proteins in aqueous solution
The high optical power provided by the laser light source enables transmission measurements using higher path lengths and protein concentrations as low as 2.5 mg mL−1
Summary
Infrared spectroscopy is a powerful and established analytical method to study the structure of proteins [1]. An experimental limitation to investigations of protein secondary structure in aqueous solutions with state-of-the-art Fourier transform infrared (FT-IR) spectrometers is constituted by the low feasible path lengths of transmission cells. This constraint originates from the combination of two factors: the high molar absorption coefficient of the HOH bending band of water near 1645 cm−1 that overlaps with the protein amide I band and the low emission power provided by the thermal light sources (globars) that are used in FT-IR spectrometers. This limitation comes along with laborious cell and sample handling as well as the need for high protein concentration (>10 mg mL−1) [2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
