UCSF

Proteins are the macromolecular complexes responsible for carrying out the majority of cellular processes ranging from signal transduction to catalyzing metabolic reactions and DNA replication. Proteins are polymers made up of amino acids that adopt complex three-dimensional shapes which are critical for their function. To better understand the structure and function of proteins structural biologist use a wide variety of experimental tools including NMR spectroscopy, X-ray crystallography, and electron microscopy (EM). Until recently, the majority of our understanding of protein structure was derived from X-ray crystallography but recent technological advances in electron microscopy have made it possible to determine the structure of a wide range of proteins to near-atomic resolution. During my Ph.D. I have primarily been focused on studying the structure and function of membrane proteins and antibody-antigen complexes using both negative stain EM and cryogenic EM (cryoEM).

The first chapter of this dissertation focuses on the utility of recombinant antibodies and their ability to probe protein structure and function in a wide variety of contexts. In particular, recombinantly expressed antibodies have a number of distinct advantages over those derived from the hybridoma approach. Of particular relevance to my thesis work I highlight the use of recombinant antibodies to study the structure of protein complexes with for single particle cryoEM.

In the second chapter I describe the structural characterization of the two-pore channel TPC1 from Arabidopsis thaliana (AtTPC1). AtTPC1 is a voltage- and ligand-gated ion channel. Previous structures of this channel revealed the first resting state of a voltage sensor in an intact ion channel. The work in this chapter expands on the previous research by mutating the luminal Ca2+ binding site shifting the open probably of the channel at 0 mV in order to chapter an activated voltage senor. Using single particle cryoEM, and a high-affinity recombinantly expressed antibody fragment, we determined two new structures of the voltage sensor. This information provided critical information about the voltage activation mechanism for ion channels and represented one of the first examples of a single voltage gated ion channel trapped in multiple conformations.

In the third chapter I present some unpublished work on a new immunoprecipitation-based method to rapidly characterize recombinant antibodies identified from phage display libraries. This method takes advantage of the minimal sample requirements of negative stain EM in order to give information about the epitope that the Fab binds as well as an estimate for the relative affinity. The technique greatly increases the rate at which it is possible to go from a panning to selecting the most useful Fabs to be used for structure determination using single particle cryoEM.