1992
DOI: 10.1073/pnas.89.24.12023
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Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

Abstract: We have used a tryptophan-requiiing Escherichia coli auxotroph to replace the three tryptophan residues of A cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved >95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-A cI are clearly distinct from A cI repressor and that the fluorescence of 5-OHTrp-A cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-… Show more

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Cited by 82 publications
(75 citation statements)
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“…There have been some qualitative experimental studies using 5-hydroxytryptophan, such as with bacteriophage λ-cI repressor, in which 5-hydroxytryptophan replaced the three wild-type tryptophans. 8 The absorption and emission spectra of the 5-hydroxy derivative had an absorption shoulder at a wavelength 30 nm longer than found for native tryptophan within the protein environment. Another study 9 looked at phosphoglycerate kinase (PGK).…”
Section: Introductionmentioning
confidence: 91%
See 1 more Smart Citation
“…There have been some qualitative experimental studies using 5-hydroxytryptophan, such as with bacteriophage λ-cI repressor, in which 5-hydroxytryptophan replaced the three wild-type tryptophans. 8 The absorption and emission spectra of the 5-hydroxy derivative had an absorption shoulder at a wavelength 30 nm longer than found for native tryptophan within the protein environment. Another study 9 looked at phosphoglycerate kinase (PGK).…”
Section: Introductionmentioning
confidence: 91%
“…[6][7][8][9] The first absorption band of 5-hydroxytryptophan is significantly longer than that of tryptophan, allowing easy discrimination of the signal. There have been some qualitative experimental studies using 5-hydroxytryptophan, such as with bacteriophage λ-cI repressor, in which 5-hydroxytryptophan replaced the three wild-type tryptophans.…”
Section: Introductionmentioning
confidence: 99%
“…Attempts to determine the percentage of 7-ATrp incorporation by amino acid analysis were not successful due to the low content of Trp (<1% of the amino acids is Trp) and coelution of other hydrolysis products with the same retention time as 7-ATrp. The limited solubility of EII mtl (micromolar concentrations) and 11 tyrosine residues in EII mtl , including a tyrosinate, prevented an estimation of the efficiency of analogue incorporation via UV spectroscopy (17). However, recording excitation spectra at different emission wavelengths provided this information.…”
Section: Resultsmentioning
confidence: 99%
“…Efficient incorporation of Trp analogues in a specific protein can be achieved when its expression is under the control of an inducible promoter as demonstrated in 1992 by Szabo and Ross (17,26). In this approach, an E. coli Trp auxotroph is grown in a Trp-containing medium.…”
Section: Discussionmentioning
confidence: 99%
“…Fluorescent markers can be introduced into ligands and proteins by covalent modifications of specific functional groups (thiols, amines) engineered by site-directed mutagenesis allowing selective, single-site labelling. Recombinant protein expression techniques using auxotroph E. coli (Ross et al, 1992) and suppressor tRNA technology can be used for site-specific introduction of unnatural fluorescent amino acids (Mendel et al, 1995) allowing selective monitoring of conformational changes in homodimers (Damian et al, 2006). Semisynthetic techniques allowing the engineering of proteins with chemically defined site-specific modification have also been increasingly used (Focke and Valiyaveetil, 2010).…”
Section: B Fluorescence Spectroscopymentioning
confidence: 99%