Azatryptophans endow proteins with intrinsic blue fluorescence.
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 approximately 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.
Max Planck Institute of Biochemistry, Molecular Biotechnology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Sandra
S
Lepthien
Michael G
MG
Hoesl
Lars
L
Merkel
Nediljko
N
Budisa
D017304:Annexin A5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001372:Aza Compounds / Q000737:chemistry*; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003116:Color; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry; D015394:Molecular Structure; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D017304:Annexin A5; D001372:Aza Compounds; D001672:Biocompatible Materials; D015335:Molecular Probes; D014364:Tryptophan