Chemical Crosslinking to Study THEMIS Protein-Protein Interactions Open Access

Abstract

Ever since its identification in 2009, THEMIS has been a focus of immunological research due to the pivotal role it plays in the positive selection and development of T cells by working with proteins Shp1 and Grb2. Scientists have long tried to characterize the protein-protein interaction of THEMIS and its two CABIT domains with Shp1 and Grb2. This project aims to investigate the viability of chemically crosslinking THEMIS to Grb2 and Shp1. After purifying THEMIS, its CABIT domains, Shp1 and Grb2, various crosslinking assays were preformed using two different chemical methods. One crosslinker, BS3 was added to the protein mixture and allowed to crosslink at lysines and other nucleophilic amino acid residues. The other crosslinking method involved incorporating a photoreactive unnatural amino acid, pBPA, at spots on CABIT1 close to the predicted binding site with Shp1. Both THEMIS and its domain CABIT2 were found to crosslink with BS3 and conformed via Western Blot analysis. Less success has been found with pBPA. Only truncated expression or no expression of CABIT1 with pBPA has been observed, so no crosslinking trials have been conducted. Overall, there is still much more investigation needed balancing reaction conditions for the expression of mutated CABIT1 and crosslinking with both pPBA and BS3. Future experimentation could elucidate a crosslinked structure of THEMIS to Shp1 or Grb2, allowing further study into T cell development to progress.

Table of Contents

Introduction 1

The Adaptive Immune System 1

THEMIS as a Regulator of T Cell Development 2

Chemical Strategies for Studying THEMIS 5

Methods 12

Protein Purification 12

BS3 Crosslinking Trials 13

Unnatural Amino Acid Incorporation Design and Preparation 15

Unnatural Amino Acid Incorporation and Expression Screens 17

Results 20

Protein Purification 20

BS3 Crosslinking Trials 23

Unnatural Amino Acid Incorporation Design and Preparation 26

Unnatural Amino Acid Incorporation and Expression Screens 29

Discussion 32

References 36

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Keyword	THEMIS chemical crosslinking adaptive immune system T-cells protein-protein interactions
Committee Chair / Thesis Advisor	Tyler Beyett, Emory University
Committee Members	Tracy McGill, Emory University Matthew Weinschenk, Emory University

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