Abstract 1568 Assessing the role of AhR and TSPO in calcium regulation in Murine mitochondria

Cataracts are the leading cause of blindness in the world, and over 20 million cataract surgeries are performed each year to restore vision.Crystallins are a class of ocular lens proteins responsible for the maintenance of lens transparency.Our research aims to understand how biologically significant mutations in Bovine Gamma B Crystallin, a homolog of Human Gamma D Crystallin, affect the temperature at which these proteins begin to aggregate, known as the cloud point.Mutations or modifications to the crystallin protein structure can lead to enhanced intermolecular interactions, resulting in aggregation and elevated cloud point temperatures; cloud points close to physiological temperatures have been implicated in cataract formation.We have expressed and purified recombinant wild-type Bovine Gamma B Crystallin, along with two point mutants: S130W and P23T.Preliminary data indicate that these mutations significantly impact the cloud point.In the case of the S130W mutant, irreversible clouding occurred at 38.8 C at a concentration of 40 mg/mL, which is slightly above physiological temperature (37 C).Additionally, we learned that homogenization of the protein sample throughout data collection is imperative to prevent aggregates from settling in order to obtain an accurate cloud point temperature.Further work is warranted to understand how these point mutations affect intermolecular interactions and contribute to clouding.