New methodology is proposed for the in vivo labeling of recombinant proteins with small-molecule probes (e.g., fluorophores, crosslinkers, photo switches, and spin labels). This methodology will employ re-engineered mutants of biotin ligase (BirA), a bacterial enzyme that site-specifically biotinylates a lysine side-chain within a 13-amino acid consensus sequence (the """"""""acceptor peptide""""""""; modified lysine residue shown in blue, below). By altering the biotin binding site of BirA to accommodate a range of biotin analogues and other small molecules, we expect to generate enzymes (""""""""probe ligases"""""""") that site-specifically conjugate various probes to recombinant proteins bearing the acceptor peptide. Protein-conjugated biotin analogues beating ketone, azide, or alkyne groups can be bio-orthogonally ligated to appropriately functionalized fluorophores or other biophysical probes via hydrazone formation, Staudinger ligation, and [3+2] Sharpless cycloaddition, respectively.
Specific aims : 1. Development of a probe ligase that works in vitro. 2. Development of a probe ligase that works in living cells. 3. Application of the methodology to identification of protein interaction partners for glutamate receptor.
|Liu, Daniel S; Nivón, Lucas G; Richter, Florian et al. (2014) Computational design of a red fluorophore ligase for site-specific protein labeling in living cells. Proc Natl Acad Sci U S A 111:E4551-9|
|Uttamapinant, Chayasith; Sanchez, Mateo I; Liu, Daniel S et al. (2013) Site-specific protein labeling using PRIME and chelation-assisted click chemistry. Nat Protoc 8:1620-34|
|Liu, Daniel S; Tangpeerachaikul, Anupong; Selvaraj, Ramajeyam et al. (2012) Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J Am Chem Soc 134:792-5|
|Uttamapinant, Chayasith; Tangpeerachaikul, Anupong; Grecian, Scott et al. (2012) Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew Chem Int Ed Engl 51:5852-6|
|Liu, Daniel S; Phipps, William S; Loh, Ken H et al. (2012) Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells. ACS Nano 6:11080-7|
|Slavoff, Sarah A; Liu, Daniel S; Cohen, Justin D et al. (2011) Imaging protein-protein interactions inside living cells via interaction-dependent fluorophore ligation. J Am Chem Soc 133:19769-76|
|Zou, Peng; Ting, Alice Y (2011) Imaging LDL receptor oligomerization during endocytosis using a co-internalization assay. ACS Chem Biol 6:308-13|
|Jin, Xin; Uttamapinant, Chayasith; Ting, Alice Y (2011) Synthesis of 7-aminocoumarin by Buchwald-Hartwig cross coupling for specific protein labeling in living cells. Chembiochem 12:65-70|
|Cohen, Justin D; Thompson, Samuel; Ting, Alice Y (2011) Structure-guided engineering of a Pacific Blue fluorophore ligase for specific protein imaging in living cells. Biochemistry 50:8221-5|
|Uttamapinant, Chayasith; White, Katharine A; Baruah, Hemanta et al. (2010) A fluorophore ligase for site-specific protein labeling inside living cells. Proc Natl Acad Sci U S A 107:10914-9|
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