Open AccessAn unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba 3 from Thermus thermophilus
Author(s) -
Liu Bin,
Luna V. Mitch,
Chen Ying,
Stout C. David,
Fee James A.
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309107054176
Subject(s) - thermus thermophilus , crystallization , protein crystallization , crystallography , mutant , cytochrome c oxidase , membrane protein , membrane , enzyme , integral membrane protein , chemistry , biochemistry , organic chemistry , escherichia coli , gene
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 ba 3 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 P 4 3 2 1 2, whereas the mutant proteins crystallize in space group P 4 1 2 1 2 with a different packing arrangement. Crystals of the P 4 3 2 1 2 form occasionally diffracted to 2.4–2.3 Å resolution following controlled dehydration, while those of the P 4 1 2 1 2 form routinely diffracted to between 3.0 and 2.6 Å for crystals that had been cryoprotected but not dehydrated.