The expression, purification, and crystallization of the HNOX regulatory domain of bovine soluble guanylate cyclase

Abstract

Soluble guanylate cyclase (sGC) converts GTP to cGMP, which plays a crucial role in vasodilation. The β subunit contains a heme NO/oxygen‐binding (HNOX) domain. NO binding to HNOX likely induces a conformational change leading to sGC activation. We aim to elucidate the mechanism of sGC activation using structural studies.Our early results revealed that the β193‐HNOX construct is poorly expressed in E. coli. To solve this problem, we made 12 truncations (from residues 187 to 198) by site‐directed mutagenesis. Cells were transformed with all 12 mutant plasmids and expression conditions were tested. We found that 3 constructs were expressed at high levels, and that only 2 proteins were soluble. Ion exchange and size exclusion chromatography yielded large amounts of partially pure samples. The UV/Vis spectra of both proteins revealed that the heme pocket is intact and that NO is bound to the heme. We are now using immunoaffinity purification to improve the sample purity and attempt crystallization.In summary, we used a systematic mutagenesis approach to obtain novel bovine HNOX sGC constructs with high levels of expression and solubility in E. coli. These constructs will be key to solve the first x‐ray structure of a mammalian HNOX domain that will provide a structural basis to understand the mechanism for NO activation.Work supported by American Heart Association Scientist Development Grant (E. Garcin)