NABBs:  Nanoscale Apolipoprotein-bound Bilayers

Summary. 
The Sakmar Laboratory recently developed a nanoparticle technology termed NABBs (nanoscale lipoprotein-bound bilayers), to study heptahelical G protein-coupled receptors (GPCRs) in a controlled artificial membrane environment.  NABBs comprise a phospholipid bilayer disc, approximately 10 nm in diameter, surrounded by a belt of 2 interlocking apolipoprotein A1 molecules.  NABBs are soluble and stable for long periods of time in solution.  Members of the Sakmar Laboratory devised a rapid method to incorporate membrane proteins, including GPCRs, into NABB particles.  NABBs that contain GPCRs can then be purified by affinity or size-exclusion chromatography and characterized by a variety of analytical methods, including electron microscopy and fluorescence imaging spectroscopy. 

Potential Impact of the NABB Technology. 
One main advantage of NABBs is that they can be adapted to serve as a membrane-mimetic structure to study integral membrane proteins in a native-like bilayer environment.  Each component of the NABB structure, including the phospholipids, zap-1 and encased GPCR, can be labeled independently.  The labels can be used to quantitate NABB components or to interrogate the structure and function of the isolated GPCR.  Once a GPCR is trapped inside a NABB particle, both surfaces of the receptor, intracellular and extracelluar, are accessible to the aqueous environment simultaneously.  This feature provides a great advantage to study the process of signal transduction across a bilayer, which occurs when a ligand binds on one side of the receptor to trigger a conformational change on the other side of the receptor.
Members of the Sakmar Laboratory incorporated the visual pigment rhodopsin into NABB particles and showed that it functioned normally.  They also showed that a single isolated rhodopsin molecule could activate G proteins in response to light and that a receptor dimer was no more efficient than a monomer in G protein activation. Recombinant expressed rhodopsin as well as expressed chemokine receptor CCR5 and the glucagon receptor, a prototypical family B GPCR, have also been incorporated into NABBs. 
For example, in recent preliminary work, members of the Sakmar Laboratory have prepared and characterized NABBs that contain functional recombinant human CCR5.  To detect correctly folded CCR5 in NABBs, a novel sandwich ELISA assay protocol in a 96-well plate format was devised where the CCR5-NABBs were sequentially sandwiched between two antibodies recognizing specific, distinct epitopes on the CCR5-NABB assembly.  In one formulation of the assay, the capture antibody, 2D7, is a monoclonal antibody (mAb) that is known to recognize a split epitope on the extracellular side of CCR5 only when they are in proximity to each other, which would be the case in a correctly folded form of CCR5.  The secondary mAb, 1D4, recognizes an engineered linear amino-acid sequence at the C-terminal tail of CCR5.  The NABB technology, when combined with the ELISA assay, can be exploited to screen for conditions that preserve heterologously expressed CCR5 in its native conformation after detergent extraction and reconstitution.