NDLI: Fluorescence Resolution of the Intrinsic Tryptophan Residues of Bovine Protein Tyrosyl Phosphatase

Content Provider	Journal of Biological Chemistry (JBC)
Author	Pokalsky, Christine Wick, Peter Harms, Etti Lytle, Fred E. Etten, Robert L. Van
Abstract	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 Å and Trp39 at least 20 Å 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 Kav and kq.
Related Links	http://www.jbc.org/content/270/8/3809.abstract
Ending Page	3815
Starting Page	3809
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	8
Volume Number	270
DOI	10.1074/jbc.270.8.3809
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1995-02-24
Access Restriction	Open
Subject Keyword	Protein Chemistry and Structure
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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