research goal and methods

The goal of the research is to compare the two mutants of the rhodopsin mutation, P23H, and S15N. Specifically, the researchers were interested in how the mutations in the N terminus domain affect their invitro biochemical properties, their UV visible absorption, Fourier transform infrared, circular dichroism and metarhodopsin II fluorescence spectroscopy properties.  While this was not a formal goal of the research, the study also found that chlorin e6have the ability to rescue mutated rhodopsin protein that had its stability affected.

In the experiment, the proteins were first prepared through PCR mutagenesis technique. The genes were then placed into HEK293S cell lines and the P23H and V15S opsins were established. The cells were harvested and the proteins were purified using immunoaffinity chromatography. 

In the first experiment, the proteins were subjected to UV spectrophotometry, which would establish the degree of misfolding of the mutated protein. For the second experiment, both of the mutant proteins had their oligosaccharide chain from rhodopsin glycosylation sites cleaved using PNGase F. The resulting immunoblot was used for densitometric analysis of the immunoblot was done using Image J, then the Box plot was used to summarize the result of the analysis. This method would establish the amount of aggregation that may occur in cells, which would disrupt their protein function. For the third experiment, MetaII fluorescence was measured for three proteins to measure the amount of aggregation that may occur in the cell. For the fourth experiment, Fourier transforms infrared spectra were collected for COS-1 cell rhodopsin to gain structural insight into the degree of misfolding in the two mutants. For the fourth experiment, circular dichroism spectra were measured with the samples of rhodopsin and Ce6 at 2.5 and 100 micro M this would indicate the stability of the presence and absence of Chlorin e6.