Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants

P. H. Axelsen, Z. Bajzer, F. G. Prendergast, P. F. Cottam, C. Ho

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Abstract

Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tryosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises >90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement.

Original languageEnglish (US)
Pages (from-to)650-659
Number of pages10
JournalBiophysical Journal
Volume60
Issue number3
StatePublished - 1991

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Tryptophan
Fluorescence
Escherichia coli
Proteins
Photons
glutamine transport proteins
Fluorescence Spectrometry
Phenylalanine
Magnetic Resonance Spectroscopy

ASJC Scopus subject areas

  • Biophysics

Cite this

Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants. / Axelsen, P. H.; Bajzer, Z.; Prendergast, F. G.; Cottam, P. F.; Ho, C.

In: Biophysical Journal, Vol. 60, No. 3, 1991, p. 650-659.

Research output: Contribution to journalArticle

Axelsen, P. H. ; Bajzer, Z. ; Prendergast, F. G. ; Cottam, P. F. ; Ho, C. / Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants. In: Biophysical Journal. 1991 ; Vol. 60, No. 3. pp. 650-659.
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