期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 42, 页码 16095-16100出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0802804105
关键词
expanded genetic code; imaging; optical probes; protein engineering and design; red-shift
资金
- BioFuture Program of the Federal Ministry of Education Research of Germany
- Munich Center for Integrated Protein Sciences (CIPSM)
Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5- Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of approximate to 130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.
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