An unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba(3) from Thermus thermophilus.

Content Provider	Europe PMC
Author	Liu, Bin Luna, V. Mitch Chen, Ying Stout, C. David Fee, James A.
Copyright Year	2007
Description	Mutation of symmetry-related (in space group P43212) glutamate and lysine residues on the water-exposed surface of an integral membrane protein, cytochrome ba 3, from T. thermophilus leads to robust crystallization of the protein in space group P41212; crystals of these mutant proteins show X-ray diffraction to molecular resolution. 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.
Related Links	https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC2344090&blobtype=pdf
Volume Number	63
PubMed Central reference number	PMC2344090
Issue Number	Pt 12
Issue Number	pt 12
PubMed reference number	18084085
Journal	Acta Crystallogr Sect F Struct Biol Cryst Commun
e-ISSN	17443091
DOI	10.1107/s1744309107054176
Language	English
Publisher	International Union of Crystallography
Publisher Date	2007-11-21
Access Restriction	Open
Rights License	© International Union of Crystallography 2007
Subject Keyword	surface engineering integral membrane proteins cytochrome c oxidase cytochrome ba3 Thermus thermophilus
Content Type	Text
Resource Type	Article
Subject	Biochemistry Condensed Matter Physics Genetics Biophysics Structural Biology