Crystallization of mitochondrial creatine kinase on negatively charged lipid layers.
Polymorphic forms of crystals of mitochondrial creatine kinase (Mi-CK) octamers were generated by the lipid-layer technique, using cardiolipin as interphase adhesion matrix. Depending on the protein and lipid concentration, different types of monolayers and 3-D stacks thereof assembled in a low ionic strength crystallization buffer. Sodium tungstate was found to promote and stabilize the crystal formation, though in-plane crystallization was also possible in the absence of tungstate. All crystal forms exhibited a p4 symmetry with lattice parameters (a = b) ranging from 10.6 to 24.0 nm and with one or four octamers per unit cell in end-on orientation. In ice-embedded crystals, which showed a molecular packing different from that of negatively stained preparations, structural features of the Mi-CK octamer were observed at a resolution of 1 nm. The crystallization process took advantage of the electrostatic interaction between negatively charged lipid head groups of cardiolipin and positive charges located at the top/bottom faces of the Mi-CK octamer. In the absence of a cardiolipin support, Mi-CK formed linear filaments from a solution of phosphotungstate by association of octamers via their top/bottom faces. When tungstate was used instead of phosphotungstate, the filaments aligned themselves into large crystalline assemblies.
Schnyder
T
T
Institute of Cell Biology, Swiss Federal Institute of Technology, Zürich.
Cyrklaff
M
M
Fuchs
K
K
Wallimann
T
T
D000818:Animals; D002308:Cardiolipins; D003402:Creatine Kinase / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D003460:Crystallization; D007527:Isoenzymes; D008081:Liposomes; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology
D002308:Cardiolipins; D007527:Isoenzymes; D008081:Liposomes; D046911:Macromolecular Substances; D003402:Creatine Kinase