Proteins are biological machines composed of a single or multiple threads of amino acids which fold into three-dimensional shapes. These shapes and surfaces directly participate in many of the molecular interactions essential to life. Some proteins exist in lipid membranes and thus have been termed membrane proteins. Membranes serve as sites of import, export, and signaling and allow for compartmentalization; all critical functions for living organisms. After decades of research throughout the 20th century, many families of membrane proteins were revealed to form transport proteins that facilitate movement of substrates across membranes. The general group of transport proteins can be further divided into two groups: active and passive transporters. Passive transport proteins have been called channels, given their observed activity of opening to allow substrate diffusion down an electrochemical gradient. Channels are diverse both in their architecture and substrates, which include ions, small molecules, and other proteins. Despite the broad importance of channels in a vast array of biology many aspects of how these machines operate at a molecular level are yet to be revealed. Determining the structures of channels, which in many cases was not technically feasible until recently, has provided key insights into the form and function of these intricate machines. Presented here are studies of two different channels: the Translocase of the Outer Mitochondrial Membrane (TOM complex) and the light-gated ion channel, ChRmine.

The first study in this thesis examines the long-awaited architecture and transport mechanisms of the major protein gate of the outer mitochondrial membrane. The high-resolution structure of the TOM complex revealed its overall organization-- including all its core subunits, multiple assemblies of the complex, and a glimpse at the pore which provides insights into mechanisms of protein transport. The subsequent study examines the first ever structures of a light-gated ion channel in a native-like membrane environment, using lipid nanodiscs and cryogenic electron-microscopy(cryo-EM). Determining cryo-EM structures of ChRmine was a technical achievement and revealed its surprising trimeric assembly, ion conduction path, and retinal binding pocket. Through analysis of the structure, we were able to tune the kinetics of ChRmine using a rational engineering approach. Lastly, the latest work on ChRmine paves the path for the determination of structures previously too dynamic to capture. These elusive structures are attractive targets because they could lay the foundation for building the next generation of tools for studying the brain.