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- W2024455955 abstract "Tryptophan phosphorescence spectroscopy is one of the most sensitive methodologies around to study the local protein structure. For proteins in buffer, the phosphorescence lifetime (τp) directly reports about the local flexibility of the site with τp ranging from (sub) milliseconds for Trp at a solvent exposed position to several seconds if the Trp side chain is embedded in a rigid protein core. Because of the long intrinsic τp of Trp, 6.5 s, the method is extremely sensitive for quenchers e.g. oxygen and its concentration needs to be reduced to a level of several nM. This extreme sensitivity for quenchers has prevented widespread use of Trp phosphorescence in molecular biology. Substitution of the indole ring with a heavy atom is expected to significantly reduce the intrinsic τp, and thus the sensitivity for quenchers. We measured intrinsic τp values of 400 ms for 6-chloroindole, 13 ms for 6- bromoindole and 38 ms for 5-bromoindole, demonstrating the potential of these Trp analogs as phosphorescence probes in proteins. A straightforward approach to produce proteins labeled with these analogs was not available. We developed before a Trp auxotrophic strain of Lactococcis lactus able to incorporate Trp analogs in proteins which E. coli Trp auxotrophs do not translate. However, this L. lactus strain was found not able to translate bromo- or cloro-substituted Trp analogs. To overcome this, the aminoacyl t-RNA Trp synthetase enzyme from L. lactus was cloned and overexpressed in L. lactus together with a single-Trp containing target protein. This approach resulted in the efficient incorporating (87-91%) of the halogenated Trp analogs and a high protein yield (25-50 mg/L culture). Details of the methodology are presented as well as the spectroscopic characterization of the chloro- and bromo-Trp containing proteins." @default.
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- W2024455955 date "2012-01-01" @default.
- W2024455955 modified "2023-09-26" @default.
- W2024455955 title "Chloro- and Bromo-Tryptophan Analogs as Phosphorescent Probes in Proteins" @default.
- W2024455955 doi "https://doi.org/10.1016/j.bpj.2011.11.1142" @default.
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