Date of Award

4-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Science

First Advisor

Cynthia V. Stauffacher

Committee Chair

Cynthia V. Stauffacher

Committee Member 1

Jason K. Lanman

Committee Member 2

Nicholas Noinaj

Abstract

ATP-binding cassette transporters comprise a large superfamily of proteins that are involved in a variety of biological phenomenon, from bacterial metabolism to cellular homeostasis, antigen-presentation, and drug resistance. These proteins are implicated in a variety of clinically relevant phenomenon, including the human diseases cystic fibrosis, macular degeneration, and cancer. Understanding their structure-function can guide therapeutics and contribute to our overall understanding of these biological phenomena.

This study focuses on understanding the motor protein of the bacterial ribose ABC transporter in the context of transport. This complex is required for the uptake of the nucleotide precursor, ribose. Using biophysical methods, we seek to better understand the protein conformational changes and dynamics that link ATP utilization with ribose uptake. We use site-directed spin-labeled electron paramagnetic resonance spectroscopy and fluorescence binding studies to understand how the motor protein interacts with ATP, and how conformational changes associated with this interaction fuel transport.

These studies reveal the motor protein, RbsA, is regulated from across the cell membrane by the availability of the transported substrate. This regulation comes from the form of a substrate-binding protein, RbsB, which binds ribose in the periplasm and delivers it to the membrane spanning permease, RbsC. Transmembrane signaling connects RbsB arrival at RbsC with inward rotations of RbsA subdomains that stimulate ATP hydrolysis. This leads to ribose delivery to the permease translocation channel. Post-hydrolysis, outward rotation of RbsA subdomains then result in the release of RbsB from the transport complex.

This process is completed by an unusual mechanism. Though the topology of RbsA predicts two ATP binding sites, and ABC transporters typically possess two equivalent ATP binding sites in their motor proteins, loss-of-function mutations have rendered one site inactive. Fluorescence binding studies reveal both sites are occupied with ATP, yet only one remains active. Thus, the conformational changes that drive active transport are driven solely by one half of the motor.

These studies provide a model for how the ribose transporter functions. As more ABC transporters, and in particular human proteins, are observed with similar half-motors, these studies may provide a general mechanism that can be applied globally to these systems. High-resolution structural information would provide a more complete picture of ribose transporter function. In collaboration with the University of Chicago, phage display experiments have identified several dozen crystallization chaperones in the form of high-affinity synthetic antibodies that will aid crystallization of ribose transporter proteins. Crystallization trials with these reagents are currently being pursued.

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