MUTATION AND CRYSTALLIZATION OF NT30 CONSTRUCT OF SOLUBLE GUANYLYL CYCLASE

picture of Feel Kang presenting his/her poster: MUTATION AND CRYSTALLIZATION OF NT30 CONSTRUCT OF SOLUBLE GUANYLYL CYCLASE

Feel Kang , Andrzej Weichsel, William R. Montfort

MUTATION AND CRYSTALLIZATION OF NT30 CONSTRUCT OF SOLUBLE GUANYLYL CYCLASE

Soluble Guanylate Cyclase (sGC) is the receptor for nitric oxide (NO), a key signaling molecule in animals for the regulation of blood pressure, wound healing and memory formation. Binding of NO to sGC stimulates the production of cGMP from substrate GTP starting a cascade of NO-dependent signaling. The subject of our studies is sGC from Manduca sexta, which has 75% sequence similarity with human sGC and similar heterodimeric architecture, but displays better stability than the human protein. The α and β subunits of the heterodimer are composed of four domains: H-NOX, PAS, coiled-coil, and catalytic domain. NT30, a construct of the sGC protein, was cloned in an attempt to increase yield and limit aggregation problem during crystallization. The α subunit of NT30 contained the PAS and coiled-coil domains with a his-tag on the N-terminus. The β subunit contained the H-NOX, PAS, and coiled-coil domains with strep-tag on the C-terminus. NT30 construct was designed based on previous NT21 construct which was expressed in milligrams per mL quantities, had proper spectral properties but failed to produce crystals due to aggregation. In order to prevent aggregation, all cysteine residues in α and β subunits of NT30 were mutated to alanine due to the hypothesis that oxidation of sulfhydryl groups caused aggregation. Unfortunately, NT30 expression in E. coli resulted in insoluble protein sequestered in inclusion bodies, suggesting that some of the cysteine residues may play a role in protein folding or heterodimer formation. Series of NT30 mutants with restored cysteine residues have been prepared in order to find the cysteine residues necessary for proper protein expression while eliminating those, which contribute to protein aggregation.    

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