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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM072670-01
Application #
6855424
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Lograsso, Philip
Project Start
2005-01-17
Project End
2009-12-31
Budget Start
2005-01-17
Budget End
2005-12-31
Support Year
1
Fiscal Year
2005
Total Cost
$299,262
Indirect Cost
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
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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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