Energy-transfer measurements on a double fluorescent labeled ribonuclease A

Abstract

Two flourescent groups were covalently attached to [bovine pancreas] RNase A: first, the .alpha.-amino group is labeled upon reaction with fluorescein isothiocyanate, and second, one of the active site histidine residues is modified by N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Among the products of these 2 successive chemical modifications, a derivative bearing one label on Lys-1 and the other label on His-119 can be isolated and characterized. Because of their spectral preperties, these 2 fluorophores, fluorescein and N-[(acetamido)ethyl]-5-naphthylamine-1-sulfonic acid, are suitable for measuring reasonance energy transfer within a single protein molecule. The efficiency of the energy transfer is close to 100% in the native state and is reduced to about 50% in the guanidine-unfolded state. This efficiency is further diminished upon reduction of the S-S bonds in denaturing conditions. The efficiency of energy transfer was determined independently from both emission and excitation spectra of the double-labeled protein, when unfolded with intact S-S bonds. The average distance between the 2 fluorescent groups can be obtained from these measurements: it increases from 20 .ANG. at most in the native state to 46 .ANG. or more in the unfolded state.