Specific Covalent Labeling of
Recombinant Protein Molecules
Inside Live Cells
B. Albert Griffin, * Stephen R. Adams, Roger Y. Tsien
Recombinant proteins containing four cysteines at the i, i
+ÿ1,ÿi +ÿ4,ÿand i +ÿ5ÿpositions of an ÿhelix were fluorescently
labeled in living cells by extracellular administration of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein.
This designed small ligand is membrane-permeant and nonfluorescent until it binds with high
affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than
does green fluorescent protein and offers greater versatility in attachment sites as well as potential
spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target
protein so that it can be singled out from the many other proteins inside live cells and fluorescently
stained by small nonfluorescent dye molecules added from outside the cells.
B. A.ÿGriffin, Department of Chemistry and Biochemistry, University of California San Diego, La
Jolla, CA 92093-0647, USA. S.ÿR.ÿAdams, Department of Pharmacology, University of California San
Diego, La Jolla, CA 92093-0647, USA. R.ÿY.ÿTsien, Department of Pharmacology, Department of
Chemistry and Biochemistry, and Howard Hughes Medical Institute, University of California San
Diego, La Jolla, CA 92093-0647, USA.
*ÿÿ Present address: Aurora Biosciences, 11010ÿTorreyana Road, San Diego, CA 92121,ÿUSA.
ÿÿ To whom correspondence should be addressed.
Attachment of fluorescent or other useful labels onto proteins has traditionally been accomplished
by in vitro chemical modification after purification (1). Green fluorescent protein (GFP) from the
jellyfish Aequorea victoria can be genetically fused with many host proteins to produce fluorescent
chimeras in situ (2, 3). However, GFP is potentially perturbative because of its size (238 amino acids),
can usually only be fused at the NH2- or COOH-terminus of the host protein, offers a limited variety
of colors, and is of no assistance for spectroscopic readouts other than fluorescence. We therefore
designed and synthesized a tight-binding pair of molecular components: a small receptor domain
composed of as few as six natural amino acids that could be genetically incorporated into proteins of
interest, and a small (<700-dalton), synthetic, membrane-permeant ligand that could be linked to
various spectroscopic probes or crosslinks. The ligand has relatively few binding sites in
nontransfected mammalian cells but binds to the designed peptide domain with a nanomolar or
lower dissociation constant. An unexpected bonus is that the ligand is nonfluorescent until it binds
its target, whereupon it becomes strongly fluorescent.
Our approach exploits the facile and reversible covalent bond formation between organoarsenicals
and pairs of thiols. Trivalent arsenic compounds bind to the paired thiol groups of proteins
containing closely spaced pairs of cysteines or the cofactor lipoic acid (4, 5). Such binding, which is
responsible for much of the toxicity of arsenic compounds, is completely reversed by small vicinal
dithiols such as 2,3-dimercaptopropanol [British anti-Lewisite (BAL)] or 1,2-ethanedithiol (EDT),
which form tighter complexes with the organoarsenical than do cellular dithiols (6, 7). If a peptide
domain could be designed with even higher affinity than that of the antidotes for an organoarsenical
ligand, the ligand could be administered in the presence of excess antidote and specifically bind the
desired peptide domain without poisoning other proteins. To achieve this unusual affinity, we
designed a peptide domain with four cysteines already organized to bind an organic molecule
containing two appropriately spaced trivalent arsenics (Fig. 1). If the distance between the two pairs of
cysteines matched the spacing between the arsenics, the two dithiol-arsenic interactions should be
highly cooperative and entropically favorable. The four cysteines were placed at the i, iÿ+ÿ1,ÿi +ÿ4,ÿand
i +ÿ5ÿpositions of an ÿhelix, so that the four thiol groups would form a parallelogram on one side of
the helix. We chose acetyl-WEAAAREACCRECCARA-amide (8) as a model peptide for in vitro tests,
on the basis of the known propensity of peptides of the form acetyl-W(EAAAR)nA-amide (9) to form
ÿhelices. |
