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Title: Characterisation of two retinal proteins, peripherin/RDs and retinaldehyde dehydrogenase type 2
Author: Idowu, Seraphina Maria
ISNI:       0000 0001 3586 1183
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2000
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Peripherin/rds (PRDS) is an integral membrane protein that is found in the outer segment of photoreceptor cells, in which dysfunctions cause a number of retinal degenerations including retinitis pigmentosa and macular degeneration. There are over 60 disease causing mutations associated with this gene, most of which are found in the lumen (L3-4) loop domain of the protein. Retinaldehyde dehydrogenase type 2 (Raldh-2) is an enzyme found in retinal pigment epithelial layer (RPE) which is required for generation of retinoic acid (RA) from retinaldehyde (RAL). RA is required for the development of many embryonic structures including the eye, heart and spinal cord and in the regulation of many genes that are involved in the development of these structures. The purpose of this study was to characterize the L3-4 loop region of PRDS through the recombinant expression of the L3-4 loop as a fusion protein with the maltose binding protein from E. coli. Moreover, some mutations known to cause retinal degenerations, R172W/G (macular degeneration and pattern dystrophy), L185P (digenic retinitis pigmentosa) and C165Y (retinitis pigmentosa) were introduced into PRDS by site directed mutagenesis. The circular dichroism spectra of wild type and mutant loop were compared, and the results were consistent with these mutations having an effect on the topology of the loop region. The aim of the expression of the Raldh-2 enzyme was to obtain a detailed study of its mechanism. The enzyme was overexpressed as a His- tagged recombinant protein in E. coli. The Raldh-2 protein sequence was modelled onto a known aldehyde dehydrogenase structure, the following conserved amino acid residues were found to be located in the active site, C301-2, S107, N169 and E399. These were all mutated to alanine and kinetic characterization of the mutant enzymes revealed that these amino acid residues are important for catalysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available