The analysis of time resolved protein fluorescence in multi-tryptophan proteins.

In the last decades, considerable progress has been made in the analysis of the fluorescence decay of proteins with more than one tryptophan. The construction of single tryptophan containing proteins has shown that the lifetimes of the wild type proteins are often the linear combinations of the family lifetimes of the contributing tryptophan residues. Additivity is not followed when energy transfer takes place among tryptophan residues or when the structure of the remaining protein is altered upon the modification. Progress has also been made in the interpretation of the value of the lifetime and the linkage with the immediate environment. Probably all the irreversible processes leading to return to the ground state have been catalogued and their rate constants are documented. Also, the process of electron transfer to the peptide carbonyl is becoming more and more documented and is linked to the rotameric state of tryptophan. Reversible excited state processes are also being considered, including reversible interconversions between rotamers. Interesting information about tryptophan and its environment comes also from anisotropy measurements for proteins in the native, the denatured and the molten globule states. Alterations of protein fluorescence due to the effects of ligand binding or side chain modifications can be analyzed via the ratio of the quantum yields of the modified protein and the reference state. Using the ratio of quantum yields and the (amplitude weighted) average lifetime, three factors can be identified: (1) a change in the apparent radiative rate constant reflecting either static quenching or an intrinsic change in the radiative properties; (2) a change in dynamic quenching; and (3) a change in the balance of the populations of the microstates or local static quenching.