edoc

A novel tool for G-protein coupled receptor stabilization and crystallization

Amer, Marwa. A novel tool for G-protein coupled receptor stabilization and crystallization. 2020, Doctoral Thesis, University of Basel, Faculty of Science.

[img]
Preview
PDF
106Mb

Official URL: http://edoc.unibas.ch/diss/DissB_13627

Downloads: Statistics Overview

Abstract

G-protein-coupled receptors are grouped into five families of membrane proteins with seven-transmembrane helix topology. Their interaction with agonists and inverse agonists alters the signaling response that regulates the diverse array of intracellular signaling cascades. Nearly one third of drugs act by binding to GPCRs and altering the intracellular signaling profile. Since GPCRs are involved in mediating cell signaling processes, they are implicated in many diseases and are the targets of numerous therapeutic drugs. More than 60% of the drugs in the market are targeting GPCRs according to only 64 known structures of GPCRS out of almost 800. Therefore, the demand of finding solving new GPCRs structure is indeed increasing in order to better develop the drug market. The production of GPCRs suitable for X-ray studies is a challenging subject in the field of structural biology due to their low natural abundance and conformational heterogeneity. Various fusion partners, including T4 lysozyme (T4L), thermostabilized apocytochrome b562 RIL (BRIL), flavodoxin, rubredoxin, and Pyrococcus abyssi glycogen synthase (PGS), that have facilitated GPCR crystallization. Interactions between fusion proteins and the GPCR can help overcome the disadvantage of the minimal polar surface area of GPCRs that is required for protein–protein packing interactions in aqueous phase, thus improving the crystallizability of GPCR fusion proteins. Since none of the known fusion proteins provides a universal solution for GPCR crystallization, designing new fusion partners or engineering currently available ones represents an effective strategy for GPCR crystallization and crystal optimization.
The major goal of this project was to develop a new stabilizing and crystallizing tool for GPCRs. The tool that was used in this study is a thermostable coiled coil that could provide the stability for the protein and the required polar surface for crystal contacts. The coild coil is a α‐helix that could pack together in a stable oligomerization domain. Coiled coils comprise between two and eight helices arranged either parallel or antiparallel to each other. Different antiparallel coiled coil candidates that possess high potential for stabilizing a GPCR were inserted into the third intracellular loop of GPCR. They were tested for initial expression. Different construct designs were also tested, and the best design was used for further experiments. The fusion protein variants overcome the hurdles associated with GPCR stabilization and crystallization. They showed a high expression level in most of the tested cell lines. The coiled-coil-fused variants were expressed in sufficient quantities for biophysical characterization and crystallization studies. The coiled coil chimeras were well expressed in a wide range of cell lines and could be solubilized in a wide range of detergents. Moreover, they showed extremely high stability and crystallization potential. Meanwhile the coiled coil maintained its ligand binding activity. We used biophysical and structural approaches to study the variants of coiled-coil-soluble domains. The structure of the coiled-coil-T4-lysozyme (CC-T4L) soluble domain was successfully solved at 2.6Å. The structure confirmed the design and the correct folding of the coiled coil as predicted. The thermostable coiled coil is a promising fusion protein for the field of GPCR structural biology. It might be interesting to test it with a wide range of different GPCRs. In addition, combining a coiled coil with other fusion candidates such as T4-lysozyme or thermostabilized cytochrome b562RIL might offer other interesting features required for GPCR crystallization.
Advisors:Abrahams, Jan Pieter and Kammerer, Richard and Schneider, Gisbert
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Nano-diffraction of Biological Specimen (Abrahams)
UniBasel Contributors:Abrahams, Jan Pieter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13627
Thesis status:Complete
Number of Pages:1 Online-Ressource
Language:English
Identification Number:
Last Modified:05 Aug 2020 04:30
Deposited On:04 Aug 2020 14:33

Repository Staff Only: item control page