Rotational freedom of tryptophan residues in proteins and peptides.

Abstract

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.