In biology, as in construction, it's all about having tools
that fit the job. Researchers at Rockefeller
University have now created a tiny tool, more than 10,000
times smaller than the diameter of a human hair, capable of encasing
single membrane proteins from living cells. The new system, which
resembles a nanoscale sushi roll, will allow investigators to
individually stimulate these key proteins with specific molecules and
signals in order to precisely define the biological reactions that
The Nanoscale Apolipoprotein Bound Bilayers (NABBs), developed
by scientists in Rockefeller's Laboratory of Molecular Biology and
Biochemistry and reported in the Journal of Molecular Biology, is a
versatile device that can likely be adapted to any of the myriad
transmembrane receptors that direct cell activity by reacting to
molecules outside the cell and activating signals inside the cell.
"Today it is impossible to know exactly what a single protein
on the surface of a cell that has thousands of other proteins is doing.
It might be acting on its own or binding to one or more other
proteins," says Thomas Sakmar, Richard M. and Isabel P. Furlaud
Professor and head of the laboratory and the study's senior
investigator. "With this tool, we can control the receptor's membrane
environment and test all possibilities of interaction with ligands,
other receptors or other proteins. It's one way to figure out how a
complex system works."
Previously, researchers studied the functions of such proteins
by investigating literally millions of them floating together in a soup
created when cell membranes are broken apart and solubilized
chemically. But this method of studying proteins is problematic, the
researchers say: The membrane protein mixtures tend to be inhomogeneous
and it is difficult - partially due to poor stability of the
isolated proteins - to purify them in their active state in
order to understand what the receptors are doing individually.
The solution, devised by Sakmar, first author Sourabh
Banerjee, a graduate student in the Tri-Institutional Chemical Biology
Program, and Thomas Huber, a postdoc, arose as the team searched for a
way to exquisitely catalogue the functions of individual
G-protein-coupled receptors (GPCRs), a large family of transmembrane
proteins that are involved in many diseases and are often the target of
medicinal agents. The structure they built was developed using a
hard-working human transport particle, the high-density lipoprotein
(HDL), as a model system. This flat, circular structure is essentially
a complex of phospholipids belted together by apolipoprotein A-I (apo
A-I) to carry cholesterol and lipids through blood to the liver.
Assuming that evolutionary forces might have already optimized
a biological solution to an engineering problem, Huber suggested using
apo A-I from zebrafish. "Based on the sequence of zebrafish apo A-I, we
thought that it may yield structurally homogeneous discs," Banerjee
says. So in their NABB, zebrafish apo A-I (known as zap1) forms a belt
that makes two layers of lipids stick together - like the seaweed that
keeps sticky rice together in sushi.
They then devised a method to trigger rapid self-assembly of
these disc-like nanoparticles from mixtures of zap1, lipids and
extracted cellular membrane proteins. "We have made it fairly
straightforward to make these structures and they form in less than an
hour," says Banerjee, who coined the term NABBs.
The team visualized individual antibody fragments bound to the
receptors with an electron microscope. And, as a proof of principle,
they experimented with rhodopsin, a prototypical GPCR. They found that
rhodopsin was remarkably stable in NABBs ? as stable as in its native
membranes. They also found that it doesn't require a dimer, or
union of two rhodopsin receptors, to produce a response - as many
scientists had argued - but that rhodopsin can be activated in its
"Each protein is very happy inside its own disc and the beauty
is that both sides of these receptors, the part that is inside the cell
and the part that is outside, are exposed to whatever you want to test
it with," Sakmar says. "That way we can use it to monitor what happens
on both sides of the cell membrane."
"This tool can be used for a wide variety of membrane
proteins," Banerjee says. "We think it will be important for
high-throughput screening for new drugs that can bind to membrane
proteins involved in disease."