Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers.

Abstract

Mitochondrial creatine kinase isolated from chicken cardiac muscle was crystallized by vapor diffusion techniques. Depending on the growth conditions, fine needles and platelets as well as large single crystals appeared after a few days. Large crystals were shown to diffract to at least 3.2 A resolution (Schnyder, T., Winkler, H., Gross, H., Sargent, D., Eppenberger, H. M., and Wallimann, T. (1990) Biophys J. 57, 420 and thus are suited for a detailed X-ray analysis in the future. The relatively high density of single crystals measured by a linear organic solvent density gradient indicates a tight packing of mitochondrial creatine kinase molecules within the crystals. Microcrystals, however, were subjected to electron optical examination either after prefixation with glutaraldehyde followed by conventional negative staining or by freeze-fracturing crystals in mother liquor and heavy metal replication with platinum/carbon. In both cases the crystals exhibited a square lattice with parameters of a = b = 139 A and a = b = 132 A in negatively stained and replicated crystals, respectively. No other lattice parameters were found, suggesting that these microcrystals represent a quasi-cubic three-dimensional lattice, which is in accordance with the finding that the building blocks of the crystals are the cube-like octamers described (Schnyder, T., Engel, A., Lustig, A., and Wallimann, T. (1988) J. Biol. Chem. 263, 16954-16962). Digital image processing applied to electron micrographs of crystals clearly revealed the arrangement of mitochondrial creatine kinase octamers in the crystal lattice as well as the subdivision of the octamer into its subdomains at a resolution of 23 A.