title: Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins.
abstract: The ability of oligomeric membrane proteins to assemble in different functional ratios of subunits is a common feature across many systems. Recombinant expression of hetero-oligomeric proteins with defined stoichiometries facilitates detailed structural and functional analyses, but remains a major challenge. Here we present two methods for overcoming this challenge: one for rapid virus titration and another for stoichiometry determination. When these methods are coupled, they allow for efficient dissection of the heteromer stoichiometry problem and optimization of homogeneous protein expression. We demonstrate the utility of the methods in a system that to date has proved resistant to atomic-scale structural study, the nicotinic acetylcholine receptor. Leveraging these two methods, we have successfully expressed, purified, and grown diffraction-quality crystals of this challenging target.
authors.affiliation: Departments of Neuroscience and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Claudio L
authors.initials: CL
authors.lastname: Morales-Perez
authors.affiliation: Departments of Neuroscience and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Noviello
authors.affiliation: Departments of Neuroscience and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: ryan.hibbs@utsouthwestern.edu.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Hibbs
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D018360:Crystallography, X-Ray / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011978:Receptors, Nicotinic