Chemistry Reference
In-Depth Information
(iii) Enzyme-linked receptors possess a catalytic site on their interior which is acti-
vated by the binding of signaling ligands in the external region. The response of
the receptor to growth factors and hormones is to trigger a guanylate cyclase or a
kinase activity. Members of the receptor tyrosine kinases include epidermal
growth factor receptor, insulin receptor, and vascular endothelial growth factor
receptor.
The pharmacology of ligand binding has recently been documented elsewhere
and is not addressed here (for a recent review see [3]). This chapter is devoted to
recent advances related to the application of single-molecule techniques to the study
of ligand interactions with membrane-bound receptors. Current progresses in
micro- and nanofabrication have led to the development of new single-molecule
methods that have proved to be valuable tools for investigating molecular
interactions in biological systems. A number of these emerging technologies will
be presented.
7.2
Labeling Methods for Cell Surface Receptors
7.2.1
General Considerations
Novel optical microscopies and spectroscopies play an important role in the elucida-
tion of biochemical interactions in live cells. In order to take full advantage, the
selective labeling of cellular components is required. Current orthogonal labeling
strategies suited for single-molecule imaging and spectroscopy of proteins and other
components in live cells or in vitro, involve the use of (i)
fluorescent analogs of
ligands, (ii)
fluorescent antibodies binding to selective epitopes on the target protein
or
fluorescent streptavidin binding to strep-tags or biotin on the target protein, (iii)
fusion to auto
uorescent proteins (AFPs), or direct labeling of proteins either (iv)
posttranslationally by chemical reactions of activated chromophores (chemical
labeling of OH, -COOH, -NH
2
or -SH comprising amino acid side chains; reversible
labeling of polyhistidine sequences with nitrilotriacetate (NTA) comprising chro-
mophores) or (v) introducing non-natural
fluorescent amino acids during protein
synthesis using the suppressor tRNA technology, and
finally (vi) posttranslational
labeling of carrier proteins fused to the protein under investigation. Each labeling
technique has particular advantages and disadvantages:
(i) Receptor ligands can be coupled to diverse high-performance organic
uoro-
phores but this is a cost- and labor-intensive process requiring a different
synthesis strategy for each particular ligand. The conjugate often has
substantially modi
ed properties when compared to the unlabeled ligand and
only allows for the study of the ligand-bound state of the receptor. Nevertheless, a
number of groups have undertaken this approach with some effort and
considerable success [4, 5].
