Fabricating Metal Nanotube-Based Biointerface for

Specific Capture of Transmembrane Proteins

 

Amit Vaish1,2, Vitalii Silin1, David Vanderah3, Klaus Gawrisch4, and Susan Krueger1

1Center for Neutron Research, 3Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland, 20899

2Materials Science and Engineering, University of Maryland, College Park, Maryland, 20742

4National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health,                       

Bethesda, Maryland, 20892

 

G-protein-coupled receptors (GPCRs) represent the largest family of membrane proteins and an important target for pharmaceuticals. GPCRs are involved in the signal transduction of a wide array of psychological processes including neuronal signaling to immune and hormonal response. Despite being such an important pharmaceutical target, only a handful of GPCR structures have been resolved, albeit in the GPCRs' inactive state by X-ray crystallography. A detailed structural and functional understanding of the GPCR is required for rational design of therapeutics tailored towards finding cures for the diseases caused by GPCR malfunctions. The key challenge facing GPCR characterization is the protein sample preparation involving receptor protein purification and reconstitution while maintaining functional activity. In order to provide an interface for selective reconstitution of the receptor protein to the surface with minimal nonspecific protein adsorption, we are employing organic synthesis strategies to design thiol-based “chemical hooks” to provide optimal control of the orientation, stability and coverage of the GPCR to the surface. Furthermore, we are fabricating highly conformal platinum nanotubes inside commercially available anodic alumina membranes using atomic layer deposition (ALD). We are developing ALD process conditions for conformal platinum coating by tuning the precursor exposure times, and subsequently monitoring the cross-sections of membranes using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The surface of metal nanotubular membranes is modified with self-assembled monolayers (SAM), followed by extrusion-assisted detergent-solubilized GPCR immobilization. Surface functionalization, protein immobilization, and protein/lipid molar ratio inside the nanopores have been analyzed by X‑ray photoelectron spectroscopy, solid-state NMR, UV-Vis spectroscopy, and fluorescence microscopy. The initial GPCR under investigation is human cannabinoid receptor, CB2, and a range of structural characterization techniques including neutron scattering and solid-state NMR will be employed to investigate the detailed structural and functional information. This nanostructured biointerface provides a large surface area for GPCR immobilization required for high-resolution NMR measurements, and enhances the neutron scattering for small-angle neutron scattering (SANS) experiments.