Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization
2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 5, e19677- p.Article in journal (Refereed) Published
Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-beta-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4 degrees C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)similar to 5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 angstrom resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.
Place, publisher, year, edition, pages
2011. Vol. 6, no 5, e19677- p.
IdentifiersURN: urn:nbn:se:su:diva-68133DOI: 10.1371/journal.pone.0019677ISI: 000290558500020OAI: oai:DiVA.org:su-68133DiVA: diva2:472081
authorCount :72012-01-032012-01-032012-01-03Bibliographically approved