Revealing the Mechanistic Diversity of the LeuT Fold: A Comparative Analysis of the Leucine Transporter and the Hydantoin Transporter using Electron Paramagnetic Resonance Spectroscopy

Abstract

This thesis has been focused on understanding structure-function relationships of transport proteins and the development of hybrid computational and spectroscopic approaches to understanding protein biology. The widespread application of Electron Paramagnetic Resonance (EPR) spectroscopy to proteins of unknown structures was hindered by the lack of a general strategy to place EPR spin label pairs in the primary sequence. Toward this end, I developed an algorithm that optimally selected spin labeling positions and verified this approach computationally and experimentally. By alleviating the experimental bottleneck associated with restraint selection, this algorithm set the stage for the efficient use of EPR restraints in computational structure determination. Using this aspect of my research to aid in experimental design, I sought to elucidate the transport mechanism of the Leucine Transporter (LeuT) to understand the processes underlying neurotransmitter transport in the human brain. LeuT is a bacterial homologue of the neurotransmitter:sodium symporter (NSS) family of transporters. To begin to understand the dynamic function of this class of proteins, I investigated the ion- and substrate-dependent conformational equilibria of LeuT using EPR. We proposed a novel model of transport that described previously unidentified inward-facing and substrate-occluded conformations. Also, this work revealed specific shifts in conformational equilibria associated with Na+ and substrate binding, which form the basis of the transport cycle. As a natural progression of the work conducted with LeuT, I was interested in understanding the nature of conformational dynamics in the LeuT Fold. In a similar analysis to that conducted in LeuT, I determined a novel transport cycle for the nucleobase precursor transporter, Mhp1, a member of the LeuT Fold class. A comparative analysis of the LeuT and Mhp1 transport mechanisms suggested a divergence in transport cycle despite structural similarity. My analysis proposed the source of this mechanistic divergence was the differing roles played by the co-transported ion Na+ and suggested that differential ion coupling mechanisms may define functional subfamilies within the LeuT Fold class.