Fluorescence resolution of the intrinsic tryptophan residues of bovine protein tyrosyl phosphatase.
Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.
Pokalsky
C
C
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Wick
P
P
Harms
E
E
Lytle
F E
FE
Van Etten
R L
RL
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D017510:Protein Folding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
D014364:Tryptophan; D017027:Protein Tyrosine Phosphatases