About the Prediction of Tryptophan Fluorescence Lifetimes and the Analysis of Fluorescence Changes in Multi-Tryptophan Proteins

Abstract

In the last five years we have studied the time resolved fluorescence of several proteins using multi-frequency phase fluorometry Each protein has taught us new aspects of tryptophan fluorescence and this manuscript describes the accumulated experience For a multitryptophan protein we consider the measured lifetimes to be amplitude-average lifetimes per family (short, medium, long) As a consequence the lifetimes of a multi-tryptophan protein can be calculated from the lifetimes of the individual tryptophan residues determined from the mutants Additivity, defined in this way, indicates that the mutations have not altered the environment and the fluorescence properties of the remaining individual tryptophan residues, and that there is no energy exchange among their excited states Non-additivity points to one or more of these complications Interacting pairs can be identified by checking additivity pairwise Alterations of protein fluorescence due to the effects of ligand binding or side chain modifications can be analyzed via the ratio of the quantum yield of the modified protein to that of the reference state This ratio of quantum yields can be factorized in three factors A first one describes the change in the apparent radiative rate constant reflecting a genuine change in the radiative properties or some static quenching. A second one describes the change in dynamic quenching and a third one identifies a change in the balance of the populations of the microstates and/or a change in local static quenching. In some cases the fluorescence rate constants of single tryptophan residues can be calculated from structural and spectral information The radiative rate constant is largely determined by the electric field in the neighborhood, and is therefore linked to the wavelength of maximum emission A pragmatic equation allows the calculation of k _r from this wavelength. In the absence of the well known side chain quenchers, the non-radiative rate constant seems to be determined by the distance between the CE3 atom of tryptophan and the carbonyl carbon of its peptide backbone In this way a quantitative prediction of the lifetime was possible for a couple of proteins.