Poster Presentation 2014 International Biophysics Congress

Structure and dynamics of membrane proteins in nanopore-confined lipid bilayers of various composition by ssNMR and spin-labeling EPR (#601)

Alex I. Smirnov 1 , Antonin Marek 1 , Maxim A. Voynov 1 , Wenxing Tang 1 , Alexander Nevzorov 1
  1. North Carolina State University, Raleigh, NC, USA

Ceramic nanochannels of anodic aluminum oxide (AAO) membranes provide unique mechanical templates for forming macroscopically aligned lipid bilayers of cylindrical shapes.  We have shown that such nanotubular bilayers retain many properties of unsupported lipid bilayers that model biological membranes. Further, lipid nanotube arrays are suitable for aligning membrane proteins for biophysical studies including high resolution solid state NMR and EPR and fluorescence.  The lipid nanotube arrays offer two principal advantages for such membrane proteins studies: 1) the structures retain high hydration level and macroscopic alignment under a broad range of pH and salt concentrations and 2) both leaflets of lipid nanotubes could be made accessible to small water soluble molecules.  These unique features open new opportunities for structure-function protein studies using physically the same sample.  Here we describe our recent progress in reconstructing both small pore-forming peptides and large membrane protein complexes, such as bacterial reaction center (RC) protein from Rhodobacter sphaeroides, into substrate-supported lipid nanotubes. Further, we demonstrate the use of this method for the determination of the structure of uniformly 15N-labeled Pf1 coat protein from bacteriophages in lipid nanotubes formed from either DMPC or DOPC/DMPG mixtures.   One-dimensional 31P NMR spectra demonstrate that both the lipids and membrane protein molecules are uniformly oriented within the nanotubes, whereas two-dimensional SAMPI4 spectra provide insight about lipid-induced conformational changes of the protein as evidenced by structural fitting of the NMR frequencies.  The spectra at 45 oC are indicative of fast uniaxial diffusion. This method is suitable for mapping phase diagrams for protein-containing lipid mixtures by NMR and EPR methods. Supported by U.S. DOE Contract DE-FG02-02ER15354 to AIS and NSF MRI 1229547 to AAN and AIS.