Oral Presentation 2014 International Biophysics Congress

Cryo-EM of membrane protein structure and function (#206)

Werner Kühlbrandt 1
  1. Max-Planck-Institute of Biophysics, Frankfurt Am Main, DE, Germany

Electron cryo-microscopy (cryo-EM) is a powerful method for investigating the structure and function of proteins and protein complexes at increasingly high resolution by single-particle analysis, electron crystallography or electron cryo-tomography (cryo-ET), providing insights into a wide range of important biological processes. The power the new direct electron detectors in cryo-EM is demonstrated by the structure of the 1.2 MDa Frh complex, which we have recently determined at 3.36 Å resolution by single-particle analysis.

Na+/H+-antiporters play crucial roles in pH and ion homeostasis in all organisms. MjNhaP1, a 36 kDa Na+/H+-antiporter, forms two-dimensional crystals. After incubating the crystals on the EM grid with buffers of different pH and NaCl concentration, we found by electron crystallo¬graphy that changes in pH in the absence of sodium resulted only in minor differences, whereas Na+ induced increasingly large, concentration-dependent helix movements. The conformational changes suggest a mechanism for Na+ binding and transport.

Cryo-ET is the method of choice for investigating large protein assemblies in situ. Using cryo-ET to study the arrangement of energy-converting membrane protein complexes in mitochondria and chloroplasts, we have shown that mito¬chondrial ATP synthase forms long rows of dimers along highly curved cristae membrane ridges. By contrast, the chloroplast ATP synthase is monomeric and confined to flat or minimally curved membrane regions. Our findings have implications for biological energy conversion under the different pH regimes in mitochondria and chloroplasts.

Finally, we used whole-cell cryo-ET to study the 10 kDa membrane protein PVAP from an archaeal virus. PVAP forms 1500 Å tall, 7-fold pyramids in the membrane of Sulfolobus target cells, which open like the buds of a flower into 1000 Å apertures for virus escape. By expres-sing recombinant PVAP in E. coli and yeast we found that the protein integrates indis¬crimina-tely into biological membranes, where it forms 7-fold pyramids. Combining cryo-ET with biochemistry and molecular biology we begin to understand how this small viral protein works as a universal membrane remodelling system.