An unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba(3) 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 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 approximately 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 P4(3)2(1)2, whereas the mutant proteins crystallize in space group P4(1)2(1)2 with a different packing arrangement. Crystals of the P4(3)2(1)2 form occasionally diffracted to 2.4-2.3 A resolution following controlled dehydration, while those of the P4(1)2(1)2 form routinely diffracted to between 3.0 and 2.6 A for crystals that had been cryoprotected but not dehydrated.
Liu
Bin
B
The Scripps Research Institute, Department of Molecular Biology, MB-8, 10550 North Torrey Pines Road, La Jolla CA 92037, USA.
Luna
V Mitch
VM
Chen
Ying
Y
Stout
C David
CD
Fee
James A
JA
D003460:Crystallization; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D003573:Cytochrome b Group; D008565:Membrane Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D010088:Oxidoreductases