TY - JOUR
T1 - Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins
AU - Morales-Perez, Claudio L.
AU - Noviello, Colleen M.
AU - Hibbs, Ryan E.
N1 - Publisher Copyright:
© 2016 Elsevier Ltd. All rights reserved.
PY - 2016/5/3
Y1 - 2016/5/3
N2 - Summary 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.
AB - Summary 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.
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U2 - 10.1016/j.str.2016.03.004
DO - 10.1016/j.str.2016.03.004
M3 - Article
C2 - 27041595
AN - SCOPUS:84962124774
SN - 0969-2126
VL - 24
SP - 797
EP - 805
JO - Structure
JF - Structure
IS - 5
ER -