Incorporation of selenomethionine into induced intracytoplasmic membrane proteins of Rhodobacter species.
Efficient multiple- or single-wavelength anomalous dispersion (MAD/SAD) techniques that use tunable X-ray sources at third-generation synchrotrons exploit the anomalous scattering of certain heavy atoms for determination of experimental phases. Development of methods for the in vivo substitution of methionine by selenomethionine (SeMet) has revolutionized the process for determination of structures of soluble proteins in recent years. Herein, we report methods for biosynthetic incorporation of SeMet into induced intracytoplasmic membrane proteins of two species of the Rhodobacter genus of purple non-sulfur photosynthetic bacteria. Amino acid analysis of a membrane protein complex that was purified to homogeneity determined that the extent of SeMet incorporation was extensive and approached quantitative replacement. Diffraction-quality crystals were obtained from SeMet-labeled membrane proteins purified from 2 l of culture. These methods augment the potential utility of photosynthetic bacteria and their inducible membrane systems for the production of foreign membrane proteins for structure determination.
Laible
Philip D
PD
Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA.
Hata
Aaron N
AN
Crawford
Adam E
AE
Hanson
Deborah K
DK
D002462:Cell Membrane / Q000378:metabolism*; D003460:Crystallization / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016992:Rhodobacter / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013057:Spectrum Analysis; D017356:Synchrotrons; D014961:X-Ray Diffraction / Q000379:methods
D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D012645:Selenomethionine