Summary. 
The terms nanoscience or nanotechnology generally refer to the study, creation, and/or manipulation of molecules or structures that are in the size range of 1 to 100 nm – for comparison a human hair is about 100,000 nm wide and a typical bacterium might be 1,000 nm across.  Ironically, to obtain structural information about a single GPCR, which is about 6 nm across at its widest point, methods are required for high-level expression and purification of large quantities of engineered recombinant protein.  Protein crystals are macroscopic structures.  The Sakmar Laboratory is interested in structural biology, but some information, like protein dynamics or kinetic “on-off” rates, is best obtained one molecule at a time.  Single molecule studies are now feasible because of advanced analytical and data collection techniques, but also because nanoscience provides the tools to isolate and interrogate individual molecules, or very small numbers of molecules in homogeneous samples, on “chips” for example.  The Sakmar Laboratory is committed to single-molecule studies of GPCR function in membranes and is developing a variety of novel enabling technologies. 

 NABBs.
NABBs (nanoscale apolipoprotein-bound bilayers) are engineered nano-structures inspired by the discoid-shaped high-density lipoprotein (HDL) particles found in human serum.  HDL particles comprise a protein belt structure that surrounds phospholipids in the form of an organized bilayer.  Members of the Sakmar Laboratory cloned and engineered a particular apolipoprotein from zebra fish that is designed to self-assemble rapidly in vitro in the presence of phospholipids to form a stable and homogenous discoidal membrane structure – a NABB.  NABB discs are about 12 nm in diameter, and although NABBs contain a membrane bilayer, they are soluble in aqueous solution and remain in solution indefinitely. 
NABBs are particularly useful for single-molecule studies of GPCRs because NABBs self-assemble in the presence of detergent-solubilized GPCRs and trap the GPCRs in the NABB bilayer.  One or two GPCRs per NABB disc can be trapped.  Once inside the NABB bilayer, GPCRs tend to function normally and remain stable.  For example, rhodopsin incorporated into NABBs can activate transducin and can be visualized using electron microscopy in combination with either direct nano-gold labeling or epitope mapping by Fab monoclonal antibody fragments.  One advantage of NABBs is that they allow simultaneous accessibility to both topological surfaces of a GPCR – inside and outside.  Another advantage is that NABBs can be used in microfluidics devices coupled to single-molecule imaging stations.  Members of the Sakmar Laboratory have now incorporated a number of GPCRs into NABBs, including rhodopsin, CCR5, CXCR4 and glucagon receptor. 
Single-Molecule Detection on Oriented-Tethered Bilayers. 
Members of the Sakmar Laboratory are implementing a novel strategy for single-molecule detection (SMD) experiments that relies on TIRF (total-internal reflectance fluorescence) imaging.  For SMD-TIRF experiments, micro-scale flow cells are fabricated to allow solutions to pass over a glass “chip.”  The “chip” tethers an oriented membrane bilayer containing a recombinant GPCR of interest.  Either the GPCR or its ligand can be labeled with appropriate fluorophores.  For single-molecule FRET (fluorescence resonance energy transfer) experiments, donor-acceptor FRET pairs can be used.  Complementary methods were sdeveloped to carry out bio-orthogonal labeling of expressed GPCRs using site-direct unnatural amino acid (UAA) mutagenesis.  In recent preliminary work, the SMD-TIRF approach was validated by measuring the binding and release of fluorescently-labeled MIP1a (the agonist ligand macrophage inhibitory protein 1) to CCR5.
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