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In the third chapter I present some unpublished work on a new immunoprecipitation-based method to rapidly characterize recombinant antibodies identified from phage display libraries.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.

I lay out the charge of the “Socratic fallacy” as originally presented in Peter Geach’s 1966 paper, and discuss more recent presentations of the challenge in the form of the "Priority of Definition" principle. I advance the thesis that Plato’s Socrates is primarily committed to a methodological principle (not an epistemological one), the Principle of Controversy. Further, I identify two attendant assumptions (M and E) that are background commitments for Plato’s Socrates. Together these explain some of the inconsistencies or tensions some commentators have suggested in early platonic epistemology, and encapsulate the method and interests of the Socrates we see in the definitional dialogues.

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca²⁺-binding site (Ca_i ²⁺), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca²⁺ to a cytoplasmic site (Ca_a ²⁺). An X-ray structure with Ca_a ²⁺ removed and a near-atomic resolution cryo-EM structure with Ca_i ²⁺ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca²⁺ ions for activation.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl₂, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.