<title>Energy transfer and specific fluorescence quenching effects in barnase, studied via multifrequency phase-fluorometry of tryptophan mutants</title>

Abstract

The relation between the three Trp residues in barnase has been characterized by computer studies of the molecular model. Trp-35 is shown to be a lone residue. However, the distance between, and the orientation of Trp-94 and Trp-71, should allow efficient energy transfer in the two directions. The overlap integrals have been calculated from the spectra of the individual Trp residues, by subtracting the spectrum of single Trp mutants from the spectra of the wild-type. A multifrequency phasefluorometric study is performed for wild-type barnase and mutant proteins. The lifetimes of the three tryptophans in the wild-type protein have been resolved. To Trp-35 a single fluorescence lifetime is attributed which varies in the different proteins between 4.3 and 4.8 ns and is pH independent between pH 5.8 and 8.9. Trp-71 and Trp-94 are considered to behave as an energy transfer couple with both forward and reverse energy transfer. To the couple two fluorescence lifetimes are attributed: 2.42 (+/- 0.2) ns and 0.74 (+/- 0.1) ns at pH 8.9, and 0.89 (+/- 0.05) ns and 0.65 (+/- 0.05) ns at pH 5.8. In the mutant Trp-94->Phe the lifetime of Trp-71 is 4.73 (+/- 0.008) ns at high and 4.70 (+/- 0.004) at low pH. In the mutant Trp-71->Tyr the lifetime of Trp-94 is 1.57 (+/- 0.03) ns at high and 0.82 (+/- 0.025) at low pH. From these lifetimes, energy transfer efficiencies can be calculated according to Porter. At pH 8.9 a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was found to be 86% for forward and 4% for reverse transfer (all +/- 2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Foerster. The Fluorescence lifetime of Trp-94, as determined in a mutant which lacks Trp-71, is found to be heavily quenched by the neighboring imidazole group of His-18. The results demonstrate the simultaneous forward and reverse energy transfer between two tryptophan residues and the quenching effect of a neighbor imidazole group.