PURIFICATION AND CRYSTALLIZATION OF D16/17 SPECTRlN REPEATS OF DYSTROPHIN
Muscular dystrophies are a group of genetic disorders characterized by progressive muscle weakness and wasting. Most of the dystrophies are due to the anomalies in dystrophin or its associated proteins. Neuronal nitric oxide synthase (nNOS) is a vasodilator localized to the sarcolemma that acts in response to the increased metabolic rates during muscle contraction. nNOS localization is impaired in most Duchenne muscular dystrophies (DMD) cases. The nNOS is localized by both dystrophin and syntrophin acting together. The D16/17 spectrin repeats is the part of dystrophin that binds to nNOS. It was found that restoring the dystrophin nNOS interaction could ameliorate the effects of dystrophy in mdx mouse (mouse model for DMD). The goal of this project was to crystallize D16/17 in order to study the structural details of this polypeptide and its binding site to nNOS. The BL21 clones of D16/17 gene with a GST tag were cultured and lysed. Affinity chromatography was used to isolate the protein with the GST tag. The tag was enzymatically cleaved using thrombin and was purified from GST and other proteinsusing ion-exchange chromatography. Highly pure protein was obtained. The solution was stored in -800 C. Optimum well conditions for crystallization was found using the PEG ION-II crystallization kit from Hampton. Three well conditions were varied to find the conditions that yield best crystals.