Site-specific fluorescent labeling of poly-histidine sequences using a metal-chelating cysteine.
Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA(2)-BM. NTA(2)-BM is a potential fluorophore for selective tagging of proteins in vivo.
Krishnan
Beena
B
Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, 710 N. Pleasant St, Amherst, MA 01003-9305, USA.
Szymanska
Aneta
A
Gierasch
Lila M
LM
D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009842:Oligopeptides / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
D001643:Bridged Bicyclo Compounds; D002614:Chelating Agents; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009842:Oligopeptides; D009942:Organometallic Compounds; D006639:Histidine; C037094:dibromobimane; D003545:Cysteine; D009571:Nitrilotriacetic Acid