Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination
Yi-Lynn Liang, Peishen Zhao, Christopher Draper-Joyce, Jo-Anne Baltos, Alisa Glukhova, Tin T Truong, Lauren T May, Arthur Christopoulos, Denise Wootten, Patrick M Sexton, Sebastian GB Furness
ACS PHARMACOLOGY & TRANSLATIONAL SCIENCE | AMER CHEMICAL SOC | Published : 2018
Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 re..View full abstract
Awarded by Monash University Ramaciotti Centre for Cryo-Electron Microscopy, National Health and Medical Research Council of Australia (NHMRC)
Awarded by NHMRC
This work was supported by the Monash University Ramaciotti Centre for Cryo-Electron Microscopy, National Health and Medical Research Council of Australia (NHMRC) project Grants APP1145420, APP1120919, and NHMRC program Grant (APP1055134). A.C., P.M.S., and D.W. are NHMRC Senior Principal Research, Principal Research, and Career Development Fellows, respectively. AG is an Australian Research Council Discovery Early Career Research Fellows. L.T.M. is an Australian Heart Foundation Future Leaders Fellow. The work was supported by the Monash University Ramaciotti Centre for Cryo-Electron Microscopy.