Generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology /

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Bibliographic Details
Author / Creator:Dominik, Pawel Kamil, author.
Imprint:2015.
Ann Arbor : ProQuest Dissertations & Theses, 2015
Description:1 electronic resource (196 pages)
Language:English
Format: E-Resource Dissertations
Local Note:School code: 0330
URL for this record:http://pi.lib.uchicago.edu/1001/cat/bib/10773197
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Other authors / contributors:University of Chicago. degree granting institution.
ISBN:9781321982527
Notes:Advisors: Anthony A. Kossiakoff Committee members: Robert J. Keenan; Shohei Koide; Ronald S. Rock.
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Dissertation Abstracts International, Volume: 76-12(E), Section: B.
English
Summary:Establishing the link between structure, dynamics, and function of membrane proteins remains elusive. Previous efforts to address many obstacles have presented a number of technical challenges because the relevant functional states of membrane proteins are transient, making it difficult to study them using high-resolution biophysical methods. Here, I describe a robust strategy for generating a class of high-performance antibody-based affinity reagents that have proven to be useful in determining the structures of relevant functional states of membrane proteins. These reagents are Fab fragments that are generated by phage display from fully synthetic libraries, and are called synthetic antibody fragments, or sABs. Previously, Kossiakoff lab and other research groups have developed phage display sorting strategies that can trap a desired conformational state of a protein antigen, or target a particular epitope on the protein surface. However, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents during sorting protocols can induce non-native conformational biases.
I sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipid-filled discoidal particles, to better mimic the native membrane environment. Nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli, such as ligands, ions and pH during phage display sorting experiments. Together, with other members in the lab, I have established and validated an improved protocol of sAB generation using more than 10 membrane protein systems of various architectures, functions and origin, and focused on characterizing in detail sABs specific to two of them: 1) Mj0480, an archaeal membrane protein of unknown function, and 2) CorA, a pentameric magnesium ion channel. Using Mj0480, I compared the nanodisc protocol with the standard method performed in detergent and characterized the influence of the membrane protein environment on the apparent affinity of sABs to their cognate antigen. Using CorA, I developed more sophisticated phage display library sorting strategies, resulting in a variety of sABs specific to either the open or closed conformation of the channel. Finally, together with collaborators, using sABs as crystallization chaperones, I obtained structural information on Mj0480 and CorA in several crystallization conditions, and used the generated sABs as probes in spectroscopic and functional studies. I believe that the newly described library sorting protocol with membrane protein in nanodiscs will provide a robust and generalizable technology platform to generate multiple high-performance synthetic antigen tools that can be used to study membrane proteins by the broader scientific community.