title: The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.
abstract: Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns.
authors.affiliation: Department of Biophysics, Institute of Molecular and Cellular Biophysics, Technische Universität München, Garching, D-85748, Germany.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Žoldák
authors.affiliation: Department of Biophysics, Faculty of Science, P. J. Šafárik University in Košice, Košice, 04154, Slovakia.|Centre for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, Košice, 04154, Slovakia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Jancura
authors.affiliation: Centre for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, Košice, 04154, Slovakia.|Department of Biochemistry, P.J. Šafárik University in Košice, Košice, 04001, Slovakia.
authors.forename: Erik
authors.initials: E
authors.lastname: Sedlák
mesh_terms: D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011506:Proteins; D014364:Tryptophan
keywords: intrinsic protein fluorescence; melting temperature; protein denaturation; tryptophan fluorescence