Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots

doi:10.1073/pnas.0705201104

Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots

*The CBR Institute for Biomedical Research, Inc., Department of Pathology, and
^†Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02215;
^‡Surface Logix, Inc., Brighton, MA 02135; and
^§Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142

Edited by Harvey Cantor, Dana–Farber Cancer Institute, Boston, MA, and approved July 12, 2007 (received for review June 1, 2007)

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.

Footnotes

^¶To whom correspondence should be addressed. E-mail: uva{at}cbr.med.harvard.edu
Author contributions: R.B. and U.H.v.A. designed research; R.B., C.V.C., P.T.S., and T.R.M. performed research; C.V.C., E.K., K.R.L., and T.A.S. contributed new reagents/analytic tools; R.B. analyzed data; and R.B. and U.H.v.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0705201104/DC1.
Abbreviations:

ICAM,

intercellular adhesion molecule;

LFA,

lymphocyte function-associated antigen;

pNPP,

p-nitrophenyl phosphonate;

QD,

quantum dot;

EG,

ethylene glycol.