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Time resolved fluorescence and phosphorescence properties of the individual tryptophan residues of barnase: Evidence for protein–protein interactions
Author(s) -
De Beuckeleer Kristel,
Volckaert Guido,
Engelborghs Yves
Publication year - 1999
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19990701)36:1<42::aid-prot4>3.0.co;2-j
Subject(s) - phosphorescence , fluorescence , barnase , tryptophan , chemistry , photochemistry , fluorescence anisotropy , quenching (fluorescence) , biophysics , biochemistry , amino acid , optics , ribonuclease , rna , physics , biology , gene
Steady‐state and time‐resolved fluorescence, as well as phosphorescence measurements, were used to resolve the luminescence properties of the three individual tryptophan residues of barnase. Assignment of the fluorescence properties was performed using single‐tryptophan‐containing mutants and the results were compared with the information available from the study of wild‐type and two‐tryptophan‐containing mutants (Willaert, Lowenthal, Sancho, Froeyen, Fersht, Engelborghs, Biochemistry 1992;31:711–716). The fluorescence and the phosphorescence emission of wild‐type barnase is dominated by Trp35, although Trp71 has the strongest intrinsic fluorescence when present alone. Fluorescence emission of these two tryptophan residues is blue‐shifted and pH‐independent. The fluorescence decay parameters of Trp94 are pH‐dependent, and an intramolecular collision frequency of 2 to 5 × 10 9 s −1 between Trp94 and His18 is calculated. Fluorescence emission of Trp94 is red‐shifted. Fluorescence anisotropy decay reveals the local mobility of the individual tryptophan residues and this result correlates well with their phosphorescence properties. Trp35 and Trp71 display a single phosphorescence lifetime, which reflects the rigidity of their environment. Surface Trp94 does not exhibit detectable phosphorescence emission. The existence of energy transfer between Trp71 and Trp94, as previously detected by fluorescence measurements, is also observed in the phosphorescence emission of barnase. Dynamic quenching causes the phosphorescence intensity to be protein‐concentration dependent. In addition, fluorescence anisotropy shows concentration dependency, and this can be described by the formation of trimers in solution. Proteins 1999;36:42–53. © 1999 Wiley‐Liss, Inc.