Single-molecule
spectroscopy of binding to receptors
When
pharmacologists want to know whether a drug binds to a
receptor, they do a so-called binding assay, where they introduce a
radio-labeled form
of the drug through a semipermeable barrier to bind to the receptor of
interest, and they detect binding by
the amount of radioactivity on the protein side of the barrier. The receptor is suspended
in the solution using detergents. This
microenvironment for the receptor is so different from its native
membrane
environment that the binding constants are highly suspect.
It also takes about an hour to probe binding this
way. We are probing binding of the receptors in
supported lipid bilayers, which can be made to mimic the membrane
environment,
and we are using single-molecule spectroscopy to acquire data on
physiological times scales. We are working with pharmacologists
to compare our binding
constants with
theirs and with other pharmacological data.
Preliminary
single-molecule data on the binding of an enkephalin (deltorphin II) to
the
delta-opioid receptor is illustrated in the series of fluorescence
images to the right.
Each image ia a few microns across. The bright spot in the center
of each image is from fluorescence
emanating from a single molecule of rhodamine
labeled enkephalin (deltorphin II)
transiently immobilized by virtue of its binding to the receptor
(delta-opioid). At t=96.7 s, the receptor is unoccupied so the
image is blank. At
t=97.0 s, a deltorphin molecule binds to the receptor and it remains
bound until 101.2 s, as indicated by the bright spot persisting through
this 4.2 s of elapsed time. These data show that single-molecule
spectroscopy is
powerful in is its ability to probe fast binding kinetics, which are
physiologically significant but are not measurable by conventional
technology. In addition, at 100.3 s, the fluorescence dims,
possibly from the enkephalin changing its position inside the binding
pocket. This transient dimming is an exciting observation, which
we have seen many times for this system, and it could provide
information about the structure and dynamics of the binding pocket.
Students
working on this project learn how to grow cells
and isolate the receptors. Students become
expert in fluorescence microscopy, chemical modification of surfaces,
molecular recognition, and general issues in drug discovery. Students interact with scientists in companies
developing array technology for
high
throughput screening, pharmacologists studying new drug targets, and
computational chemists investigating memrbane protein structure.
