This project employs tools of Biophysics to answer fundamental questions about the structure and function of the gated membrane channel, VDAC or mitochondrial porin, which is formed by a 31-kD protein in the mitochondrial outer membrane. The main technique used is high-resolution electron microscopy and computer-based image analysis. The channel spontaneously forms planar crystalline arrays when the outer mitochondrial membrane of the fungus Neurospora crassa is treated with phospholipase A2. The image processing technique of correlation averaging is used to obtain projection images with resolution approaching 10 angstroms from these two-dimensional crystals embedded in either vitreous ice or the sugar, aurothioglucose. Three-dimensional structures are obtained by combining projection data from variously tilted crystals. The phospholipase-induced crystals are polymorphic, i.e. there are several types, and the occurrence of certain types correlates with exposure to effectors of the channel's gating properties. Thus, it is hoped that comparison of the channel structures in the different crystal polymorphs will shed light on the conformational changes underlying gating. In order to overcome the 10 angstrom resolution limit, needed to directly visualize protein secondary structure, attempts to reconstitute the channel protein into larger, better ordered two-dimensional crystal are to be undertaken. Also, three-dimensional crysal trails will be initiated using protein overexpressed in bacteria. If successful, this will open the way for x-ray crystallographic studies. In the meantime, complementary information about the folding of the channel polypeptide will be provided by circular dichroism studies and by immunolabelling of the crystals with Fab fragments of sequence-specific, anti-peptide antibodies. %%% The cell and its internal compartments are bounded by membranes composed of lipids and proteins. The lipids form a bilayer sheath that is impenetrable to polar ions and molecules. Proteins make it possible for the different compartments to communicate, i.e., exchange material. For example, some proteins form channels, water-filled passageways across the membrane bilayer which open and close (or gate) in response to different stimuli. The basis structure of channels and the molecular mechanism(s) of their gating are only poorly understood. In this project, we are studying a channel that occurs on the surface of the intracellular organelle that is the cell's powerplant, the mitochondrion. This channel is switched by transmembrane electrical potentials between states with different degrees of openness, and this gating appears to be controlled by other proteins in the mitochondrion. Using electron crystallography and other biophysical and biochemical approaches, we are determining the three-dimensional structure of the channel in different states, in order to understand how it is formed how it opens and closes.
