title: Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.
abstract: The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3-4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison 53706.
authors.forename: C A
authors.initials: CA
authors.lastname: Royer
mesh_terms: D001426:Bacterial Proteins; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins