title: Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence.
abstract: Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues. The spectrum is dominated by the contribution of Trp-35. The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75. This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989). This histidine residue is close to Trp-94. The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction. The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein. Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94.
authors.affiliation: MRC Unit for Protein Function and Design, Cambridge IRC for Protein Engineering, Department of Chemistry, University of Cambridge, U.K.
authors.forename: R
authors.initials: R
authors.lastname: Loewenthal
authors.forename: J
authors.initials: J
authors.lastname: Sancho
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004563:Electrochemistry; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D006639:Histidine; D014364:Tryptophan; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease