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Characterisation of Rhodopsin Retinitis Pigmentosa mutants located in Intradiscal Loop 1

Neaves, Luke Christopher (2020) Characterisation of Rhodopsin Retinitis Pigmentosa mutants located in Intradiscal Loop 1. Masters thesis, University of Essex.

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Abstract

Retinitis Pigmentosa (RP) is a genetic condition that results in blindness. There are several hundred RP mutations associated with rhodopsin, a photosensitive GPCR pigment found within rod cells of the retina. Previous studies have shown that many rhodopsin RP mutants fold incorrectly or affect protein stability. This study investigates the effect of three RP mutants located in Extracellular loop 1 of bovine rhodopsin. Expression levels and protein folding have been examined using HEK 293 GnTI- cells and two methods have been used in an attempt to correct protein folding. The effects of these two correction methods on three mutations have been analysed by investigating their UV-visible absorption spectral properties, photobleaching properties, active light-activated state stability kinetics and pigment thermal stability. Pharmacological chaperone rescue with 9-cis-retinal was found to enhance expression levels and folding in two of the three mutant pigments. However, these rescued pigments were unstable at high temperatures (55 °C) when purified. The second approach involved the insertion of a disulphide bridge linking the N-terminal cap to ECL 3 and is known to increase the thermostability of WT rhodopsin. The N2C/D282C bond was found to repair two of the three mutant pigments and enhance stability, signal transduction, photobleaching and Meta-II stability to near WT levels. These two methods, when deployed together, had little additive effect. The main conclusion is that the N-terminal cap of rhodopsin is prevented from either folding correctly or docking correctly to ECL 3 due to the mutations located in ECL 1. However, when the N-terminal cap of rhodopsin is tethered in place by a disulphide bridge or when the retinal binding pocket is occupied, thermal stability and active state properties are restored in some rhodopsin RP mutants.

Item Type: Thesis (Masters)
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Luke Neaves
Date Deposited: 24 Jul 2020 08:20
Last Modified: 24 Jul 2020 08:20
URI: http://repository.essex.ac.uk/id/eprint/28298

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