TY - JOUR
T1 - Site-specific orthogonal labeling of the carboxy terminus of α-tubulin
AU - Banerjee, Abhijit
AU - Panosian, Timothy D.
AU - Mukherjee, Kamalika
AU - Ravindra, Rudravajhala
AU - Gal, Susannah
AU - Sackett, Dan L.
AU - Bane, Susan
PY - 2010/8/20
Y1 - 2010/8/20
N2 - A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.
AB - A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.
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U2 - 10.1021/cb100060v
DO - 10.1021/cb100060v
M3 - Article
C2 - 20545322
AN - SCOPUS:77955855618
SN - 1554-8929
VL - 5
SP - 777
EP - 785
JO - ACS chemical biology
JF - ACS chemical biology
IS - 8
ER -