Download Statistics
Download Statistics
Grime, Rachael L 
ORCID: https://orcid.org/0000-0002-6716-2878
(2021).
Investigating G-protein-coupled receptor structure and function using novel nano-encapsulation strategies.
University of Birmingham.
Ph.D.
|
Grime2021PhD.pdf
Text - Accepted Version Available under License All rights reserved. Download (36MB) | Preview |
Abstract
Nano-encapsulation of membrane proteins using the archetypical polymer poly(styrene-co-maleic acid) (SMA) is well established. In this study, fluorescence correlation spectroscopy (FCS) was employed to characterise binding at the G-protein-coupled receptor (GPCR), adenosine 2A receptor, solubilised with SMA to form SMA lipid particles (SMALPs). Optimal conditions were systematically ascertained and FCS was validated for the biophysical study of GPCR-SMALPs. A novel fluorescent parathyroid hormone 1 receptor (PTH1R) agonist was characterised and is ready for use in fluorescent studies with purified PTH1R-SMALP.
The rapid adoption of nanodisc technology has resulted in demand for a ‘polymer tool kit’ to provide alternatives where SMA presents limitations. Emerging polymers, poly(styrene-co-maleimide) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) were used alongside SMA to solubilise rhodopsin from rod outer segments (ROS) and characterised. SMA, SMI and DIBMA were efficient at extracting rhodopsin from ROS. Isomerisation of 11-cis-retinal was evident upon photobleaching, however, SMA and SMI restrained rhodopsin in inactive meta I conformation, whereas DIBMA supported transition to fully-active meta II.
Finally, the role of extracellular loop one (ECL1) of calcitonin receptor-like receptor was probed via systematic residue deletion. A functional role was established for several ECL1 residues and loop length, with differences between the calcitonin gene-related peptide receptor and adrenomedullin 1 receptor. This study provides insight into GPCR pharmacology and the utility of SMA-like polymers.
| Type of Work: | Thesis (Doctorates > Ph.D.) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Award Type: | Doctorates > Ph.D. | |||||||||
| Supervisor(s): |
|
|||||||||
| Licence: | All rights reserved | |||||||||
| College/Faculty: | Colleges > College of Life & Environmental Sciences | |||||||||
| School or Department: | School of Biosciences | |||||||||
| Funders: | Biotechnology and Biological Sciences Research Council | |||||||||
| Subjects: | Q Science > Q Science (General) Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology |
|||||||||
| URI: | http://etheses.bham.ac.uk/id/eprint/12240 |
Actions
 |
Request a Correction |
 |
View Item |
Downloads
Downloads per month over past year