Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.
Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
Marta
M
Fernández-Suárez
Hemanta
H
Baruah
Laura
L
Martínez-Hernández
Kathleen T
KT
Xie
Jeremy M
JM
Baskin
Carolyn R
CR
Bertozzi
Alice Y
AY
Ting
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017930:Genes, Reporter / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008025:Ligases / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D015202:Protein Engineering / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D008063:Thioctic Acid / Q000378:metabolism*
D008063:Thioctic Acid; D008025:Ligases