Energy-transfer measurements on a double fluorescent labeled ribonuclease A.
Two fluorescent groups have been covalently attached to ribonuclease 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 two 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 properties, these two fluorophores, fluorescein and N-[(acetamido)ethyl]-5-naphthylamine-1-sulfonic acid, are suitable for measuring resonance 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 disulfide bonds in denaturing conditions. The efficiency of energy transfer has been determined independently from both emission and excitation spectra of the double-labeled protein, when unfolded with intact disulfide bonds. The average distance between the two fluorescent groups can be obtained from these measurements: it increases from 20 A at most in the native state to 46 A or more in the unfolded state.
D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006639:Histidine; D007700:Kinetics; D008433:Mathematics; D009282:Naphthalenesulfonates; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D013861:Thiocyanates; C006422:1,8-I-AEDANS; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D016650:Fluorescein-5-isothiocyanate