| All eukaryotic cells require an efficient exchange of metabolites between the mitochondria and the rest of the cell. This exchange is mediated by the most abundant protein in the outer mitochondrial membrane, the Voltage-Dependent Anion Channel (VDAC), which serves as the primary pathway for the exchange of ions and metabolites between the cytoplasm and the inter-membrane space. Additionally, VDAC acts as a scaffold for proteins that modulate mitochondrial permeability and has been implicated in mitochondria-dependent cell death. Because of its critical role in mitochondrial biology, the primary objective of my Ph.D. was to obtain the crystal structure of VDAC as a first step to understanding its gating mechanism, in vivo regulation and interaction with other proteins.;X-ray crystal structures of membrane proteins are notoriously difficult to obtain. To emphasize this point, membrane proteins represent ∼30% of all proteins in each of the sequenced genomes, yet comprise less than 1% of all deposited structures in the Protein Data Bank. The reason for this discrepancy stems from the hydrophobic nature of membrane proteins, which reside in phospholipids bilayers, making them difficult to express, purify and crystallize.;To overcome these challenges associated with membrane protein crystallography, the first step was to obtain "crystallization-grade" protein that was pure, homogeneous, functional, and in sufficient quantities to set up crystallization trials. Expression, refolding and purification protocols were optimized until murine VDAC1 (mVDAC1) protein having the above properties was obtained. Purified mVDAC1 protein was extensively characterized using biochemical and biophysical studies to ensure that its identity, fold and functional properties are the same as the native protein.;The next important challenge was to obtain crystals of purified mVDAC1 protein. When crystallization using the traditional detergent-based approach did not succeed in producing high quality crystals, the bicelle method was employed and upon optimization, resulted in mVDAC1 crystals that diffracted to 2.3A resolution. In contrast to detergent micelles, bicelles are small bilayer-like discs that more closely mimic the native lipid environment. As a result, the structure we obtained likely represents that of the endogenous channel.;The final critical challenge associated with membrane protein crystallography is the phase problem. Since all other methods to determine mVDAC1 phases failed, a novel method, cysteine scanning mutagenesis, was successfully used. This method, which has also been used for LacY and vSGLT, involves using an engineered cysteine to incorporate a single mercury atom.;The mVDAC1 structure presented here represents the first high-resolution crystal structure of a eukaryotic beta-barrel membrane protein. Our findings report a detailed high-resolution structure of a new class of beta-barrel membrane proteins formed by a 19 stranded beta-sheet. The N-terminus is located inside the barrel and forms an alpha-helix halfway down the pore resulting in a partial narrowing at the center. This orientation of the helix is ideally suited to gate metabolite flux through the channel. In addition to providing concise structural details that can aid drug design, the high-resolution structure has lead to new hypothesis regarding the gating mechanisms for metabolites and ions passing through this channel providing critical information for our understanding of VDAC's role in mitochondrial physiology. |
