An unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba₃ from Thermus thermophilus

Past work has shown that it is feasible to mutate surface residues of soluble proteins and to a lesser extent membrane proteins in order to improve their crystallization behavior. Described here is a successful application of this approach to the integral membrane protein Thermus thermophilus cytochrome ba₃ oxidase. Two mutant forms of this enzyme (I-K258R and I-K258R/II-E4Q) were created in which symmetrical crystal contacts within crystals of wild-type enzyme were modified. These mutant proteins had greatly shortened crystallization times, decreasing from ∼30 d for the wild type to 1–3 d for the mutants, and crystallization was highly reproducible. Native-like proteins crystallize in space group P4₃2₁2, whereas the mutant proteins crystallize in space group P4₁2₁2 with a different packing arrangement. Crystals of the P4₃2₁2 form occasionally diffracted to 2.4–2.3 Å resolution following controlled dehydration, while those of the P4₁2₁2 form routinely diffracted to between 3.0 and 2.6 Å for crystals that had been cryoprotected but not dehydrated.