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 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 A crystal structure of 8R-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 8R-LOX in which the corresponding membrane-insertion loop is absent (Delta41-45: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 A improvement in the resolution of the diffraction data.
Neau
David B
DB
Center for Advanced Microstructures and Devices, Louisiana State University, Baton Rouge, LA 70803, USA.
Gilbert
Nathaniel C
NC
Bartlett
Sue G
SG
Dassey
Adam
A
Newcomer
Marcia E
ME
D000818:Animals; D037421:Anthozoa / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000737:chemistry*
D008565:Membrane Proteins; D008084:Lipoxygenase