Improving protein crystal quality by selective removal of a Ca 2+ ‐dependent membrane‐insertion loop

Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane‐embedded fatty acids. A Ca 2+ ‐dependent membrane‐binding function was localized to the amino‐terminal C2‐like domain of 8 R ‐lipoxygenase (8 R ‐LOX) from the soft coral Plexaura homomalla . The 3.2 Å crystal structure of 8 R ‐LOX and spectroscopic data suggested that Ca 2+ stabilizes two membrane‐insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X‐ray data. A deletion mutant of 8 R ‐LOX in which the corresponding membrane‐insertion loop is absent (Δ41–45:GSLOX) was engineered. Removal of the membrane‐insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 Å improvement in the resolution of the diffraction data.