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protein part 1

Submitted by ziweiwang on Thu, 12/05/2019 - 21:45

Mutation of RP4 mostly affects the rhodopsin protein. The structure of the protein is a protein made of an n tail, c tail, 8 alpha-helix, and 5 turns and the protein is embedded in a membrane. The normal function of the protein is to activate the G protein to start a phototransduction cascade. When the light hits Rhodopsin, retinal that is attached to the protein isomerizes and causes rhodopsin to activate the G-protein, which in turn starts the phototransduction cascade.
 An experiment that aimed at testing changes that leads to more instability in rhodopsin structure causes severe disease in patients. Specifically, the experiment aimed at testing the function of the protein that has the P23H mutation and the N15S mutation. In the first experiment, A purified protein of the mutant and wt rhodopsin were recorded using a UV spectrometer, and a thermal denaturation experiment was done on the proteins. The result showed that P23H had a lower yield of correctly folded protein compared to N15S, and the thermal stability of P23H was75% less compared to the wild type protein. This indicates that the P23H is a less stable protein compared to N15S and wt. The mutation is a more serious mutation compared to N15S indicating that people with the P23H will have serious symptoms compared to people who have an N15S mutation. In the second experiment that was done, Meta II fluorescence was measured, and the data were analyzed using a sigma plot. The result showed that Total fluorescence after light activation was lower than wt for both P23H and N15S due to aggregation. The results imply that both of the mutations were more prone to aggregation compared to the wt. In the third experiment that was done, the protein was reacted with n-glycosidase F. An immunoblot was run and used for densitometric analysis linear regression was performed on the result of the immunoblot. The result showed that the proportion of unglycosylated species was much higher in P23H than in N15S and WT and lower intensity at lower molecular weight bands indicating that there is some degradation of P23H. P23H and N15S both have a higher intensity of Dimer and higher molecular weight which indicates that there was a higher rate of aggregation. The degree of aggregation and glycosylation was higher in P23H compared to N15S which was in turn higher what wt. This shows the differences in aggregation and glycosylation explains why the severity of the phenotype differs between the two mutations.