Purification, crystallization and preliminary X-ray analysis of a mu-like calpain.
The X-ray structure of m-calpain in the absence of Ca(2+) has been described, but it has not been possible to obtain sufficient mu-calpain for structure determination. Comparison of the two structures is of interest in attempting to understand their different Ca(2+) requirements. Here, the crystallization in the absence of Ca(2+) of an inactive mutant hybrid calpain (MW approximately 100 kDa), which contains 85% of the rat mu-calpain sequence and is well expressed in Escherichia coli, is described. The properties of this calpain in its active form and particularly its Ca(2+) requirement are close to those expected for wild-type mu-calpain. Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (M(r) approximately 6000) as precipitating agent in the presence of detergent. The crystals diffract to a resolution of 2.7 A at a synchrotron source. The space group is P2(1), with unit-cell parameters a = 72.7, b = 184.6, c = 86.3 A, beta = 100.7 degrees. There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%.
Pal
Gour P
GP
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
DeVeyra
Teresa
T
Elce
John S
JS
Jia
Zongchao
Z
D000818:Animals; D002154:Calpain / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D002154:Calpain; C449002:mu-calpain