Abstract
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 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.
Original language | English (US) |
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Pages (from-to) | 1029-1034 |
Number of pages | 6 |
Journal | Acta Crystallographica Section F: Structural Biology and Crystallization Communications |
Volume | 63 |
Issue number | 12 |
DOIs | |
State | Published - Nov 30 2007 |
Externally published | Yes |
Keywords
- Cytochrome ba
- Cytochrome c oxidase
- Integral membrane proteins
- Surface engineering
- Thermus thermophilus