Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical & Biochemical Engineering

First Advisor

Hubert Yin

Second Advisor

Theodore Randolph

Third Advisor

Amy Palmer

Fourth Advisor

Joel Kaar

Fifth Advisor

Stephanie Bryant

Abstract

Membrane proteins account for approximately 30% of the human proteome, are the therapeutic target of nearly 60% of current pharmacological agents, yet account for <1% of all solved structures. This lack of information on membrane proteins extends from the difficulty in studying transmembrane domains due to their inherent insolubility and instability outside of membrane mimetics. New methods to study these domains are critical for advancing the understanding of membrane protein signal transduction.

The Toll-like receptor (TLR) family of membrane proteins serve as critical sensors of infection for the innate immune system. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for TLRs. Using a biochemical and biophysical approach in membrane mimetic systems, we were the first to report the role of isolated transmembrane domain interactions in TLR oligomerization. We showed that all TLR transmembrane domains were capable of homotypic interactions. We also demonstrated that the TLR2 transmembrane domain preferentially interacted with the transmembrane domains of its heterotypic signaling partners, TLR1 and TLR6.

To better understand the role of transmembrane domain interactions in biologically relevant membranes, we utilized acceptor photobleaching Förster resonant energy transfer to image the involvement of individual domains in TLR assembly in live cells. This technique is a simple, rapid approach to screen for both homotypic and heterotypic interactions in native protein environments. Results suggest that TLR2-TLR1 and TLR2-TLR6 transmembrane domains are interacting heterotypically in native cell membranes.

To better screen TLR signaling pathways for novel therapeutic intervention, we established a genetic reporter in native immune cells, monocytes and T-cells, for monitoring TLR signaling activity. Monocytes specifically responded to TLR2 agonists and T-cells were specific for TLR5 agonists. Utilization of these cell lines allowed for screening of transmembrane domain libraries to investigate the potential of regulating TLR2 and TLR5 signaling through transmembrane domain interactions. Furthermore, they allowed for validation of small molecule therapies to treat TLR related diseases.

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