Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072670-05
Application #
7545931
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Fabian, Miles
Project Start
2005-01-17
Project End
2009-12-31
Budget Start
2009-01-01
Budget End
2009-12-31
Support Year
5
Fiscal Year
2009
Total Cost
$282,972
Indirect Cost

  Institution

Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139

  Publications

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

Showing the most recent 10 out of 21 publications