Date

2014

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Chemical Engineering

First Adviser

Berger, Bryan W.

Other advisers/committee members

Jagota, Anand; Snyder, Mark A.; Glover, Kerney J.

Abstract

Membrane proteins comprise of 50% of all pharmaceutical targets in the human genome. These proteins reside in equilibrium between resting and active states that are responsible for various intracellular signaling. We are interested in studying a type of membrane protein belonging to the G-protein coupled receptor (GPCR) family named calcitonin receptor-like receptor (CLR). When associated with a single pass membrane protein called receptor activity modifying protein 1 (RAMP1), the CLR-RAMP1 complex forms a specific receptor for calcitonin gene-related peptide (CGRP). Dysregulation of this receptor complex is linked to various disease states including migraine and acute coronary syndrome (ACS). Small molecule antagonists targeting CLR-RAMP1 complex have been shown to be effective in treating acute migraines, however, they are only limited to intravenous delivery. Therefore, it is very important to obtain a detail understanding on how these receptors interact to form the functional receptor for superior drug design. Membrane proteins are extremely difficult to study due to their water insoluble nature. To overcome this limitation, we developed several tools to assist our research. One important mechanism that regulates membrane protein activation is receptor oligomerization. In order to gain an understanding and identify residues that govern receptor oligomerization in a straightforward and high throughput fashion, we developed E. coli transcription factor AraC-based methods named AraTM and DN-AraTM which look at receptor homo- and heterodimerization respectively. By using AraTM assay, we were able to identify a specific juxtamembrane region within the cytosolic domain (A375-P394) of receptor for advanced glycation endproducts (RAGE) which mediates its homodimerization. Moreover, we also developed a T7-based expression vector (pOmpF) using an engineered fragment of outer membrane protein F (OmpF) as the fusion protein to direct full-length membrane protein overexpression in E. coli for high-resolution structure determination. Utilizing pOmpF vector, we successfully purified thermally stable RAMP1 protein in detergent Fos-choline 15 (FC15). By using circular dichroism, dynamic light scattering, and tryptophan fluorescence spectroscopy, we were able to show that the purified RAMP1 protein has a native tertiary structure consisting of disulfide bonds with 90% helical content. Finally, using sequence-directed searches of transmembrane structural databases, we identified a P-x-x-x-T motif interface that is conserved in RAMP1 among different species as well as between different human RAMPs. By applying cAMP signaling assay, in vivo bioluminescent resonance energy transfer assay, and zebrafish RAMP1 phenotypic knockdown and rescue experiments, we were able to show that this predicted P-x-x-x-T motif plays a critical role in CLR-RAMP1 association and function. Altogether, this work not only provides innovative tools to improve membrane protein research but also sheds light on understanding the structural basis for CLR-RAMP1 receptor signaling.

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