Engineering Perfringolysin O Mutants with Different Cholesterol Binding Thresholds
Mote, Francisca Sefakor
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Cholesterol is a sterol molecule that is a primary constituent of mammalian cells. It is important for controlling membrane fluidity, stability and organization. It is also precursor for the biosynthesis of steroid hormones and bile acids. Abnormal levels of cholesterol have been implicated in some cardiac and brain conditions such as coronary heart disease. Perfringolysin O (PFO) is a water-soluble toxin, whose binding and insertion into cell membranes requires cholesterol. Cholesterol activity in a membrane is related to the ability of cholesterol to interact with molecules at the membrane surface. The binding of PFO is thought to be dependent on cholesterol activity and requires high cholesterol levels on the membrane’s surface for binding. Currently, cholesterol probes, such as filipin, measure overall cholesterol concentration whereas PFO measures cholesterol activity. The aim of this project is to design a probe using PFO that would detect a wide range of cholesterol activity levels. Based on previous research, we hypothesized that hydrophobic mutations in the non-conserved regions of PFO would allow the protein to interact better with the hydrophobic membrane core, and thus require less cholesterol. The additive effect of two previously identified mutants, D434S and A437V was also determined. A401L and A401F required less cholesterol compared to native PFO. The double mutant did not display an additive effect.