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Fluorescence lifetime distributions report on protein destabilisation in quenching experiments

Paper ID Volume ID Publish Year Pages File Format Full-Text
30190 44463 2013 7 PDF Available
Title
Fluorescence lifetime distributions report on protein destabilisation in quenching experiments
Abstract

•We did acrylamide quenching experiments on 7 proteins to identify acrylamide’s undesired effect on the protein structure.•We measured acrylamide and GuHCl concentration dependence of the FWHM of fluorescence lifetime distributions.•We also provided a direct correlation between the acrylamide effect and denaturation using CD spectroscopy.•We found that acrylamide has perturbed the conformational states of the investigated proteins.•FWHM of the lifetime distribution is a powerful analytical tool to identify the undesired effects of acrylamide quenching.

Tryptophan is the most often investigated intrinsic fluorophore due to its abundance in proteins and its sensitivity to different environmental conditions. Fluorescence quenching is a powerful method to study proteins and acrylamide is a frequently applied quencher in these investigations. Quenching experiments are sometimes distorted by the undesired protein–quencher interactions that can result in a misinterpretation of the results. Here we focused on the identification of the possible side-effects of acrylamide applying fluorescence lifetime measurements. To provide reference data for protein denaturation the fluorescence parameters were also recorded in the presence of different concentrations of guanidine hydrochloride. In circular dichroism experiments we characterized directly the acrylamide effect on the tertiary structure of the proteins. According to the obtained data in experiments with seven tryptophan-containing proteins the full width at half maximum (FWHM) of the fluorescence lifetime distribution is an appropriate parameter to monitor the undesired effects of acrylamide on the proteins.

Keywords
Tryptophan; Protein stability; Fluorescence quenching; Fluorescence lifetime distribution; Circular dichroism; Protein denaturation
First Page Preview
Fluorescence lifetime distributions report on protein destabilisation in quenching experiments
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 129, 5 December 2013, Pages 108–114
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering