title: Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots.
abstract: The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in vivo.
authors.affiliation: The CBR Institute for Biomedical Research, Inc., Department of Pathology, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Bonasio
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Carman
authors.forename: Enoch
authors.initials: E
authors.lastname: Kim
authors.forename: Peter T
authors.initials: PT
authors.lastname: Sage
authors.forename: Kerry R
authors.initials: KR
authors.lastname: Love
authors.forename: Thorsten R
authors.initials: TR
authors.lastname: Mempel
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
authors.forename: Ulrich H
authors.initials: UH
authors.lastname: von Andrian
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009596:Nitrophenols; D063065:Organophosphonates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D045663:Quantum Dots; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity