Membrane Protein Purification and Crystallization

Abstract

Cytochrome c oxidase, the terminal complex of the respiratory chain of mitochondria and many bacteria, catalyzes the electron transfer from cytochrome c to molecular oxygen, thereby reducing the latter to water. Thermus thermophilus HB8 (ATCC 27634) is an extremely thermophilic gram negative bacterium. It grows at temperatures between 47° and 85°C. Depending on the fermentation conditions and the 02-supply, this organism expresses two different oxidases known as the caa3-and the ba3-type. This chapter describes a new procedure for the preparation of monodispersed highly active ba3-type cytochrome c oxidase from Thermus thermophilus. The enzyme is isolated from the membrane using Triton X-100 and purified by subsequent anion-exchange chromatographies first in Triton X-100 and then in dodecyl-β-D-maltoside. The best crystals were grown in nonyl-β-D-glucoside using PEG 2000 as precipitant. However, these crystals show drastic changes in their cell constants if measured in a standard capillary and a relatively weak diffraction power in their native (as grown) state. In order to obtain a useful dataset, it was necessary to transform these crystals by dehydration.