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| Peptide Synthesis | Cell Reports 30, 1–11, March 17, 2020 11 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies FLAG M2 antibody Millipore-Sigma Cat# F3165; RRID: AB_259529 Gaq/11 antibody Millipore-Sigma Cat# G4290; RRID: AB_259903 Bacterial and Virus Strains JM109(DE3) Promega Cat# P9801 Rosetta 2(DE3) DH5a T1R Sigma Cat# 71400 Invitrogen 12297016 Chemicals, Peptides, and Recombinant Proteins Benzamidine Sigma Cat# L2884 Leupeptin Sigma Cat# L2884 CHAPS Anatrace Cat# C316 GDP Sigma Cat# 7127 ESF921 culture medium Expression Systems Cat# 96-001 TCEP [3H]-GDP Sigma Cat# C4706 PerkinElmer Cat# NET966250UC IPTG Goldbio Cat# I2481 Myristic acid Sigma Cat# M3128 Lysozyme Sigma Cat# 62970 Chelating Sepharose Fast Flow GE Healthcare Cat# 17057502 DTT Goldbio Cat# DTT Apyrase NEB Cat# M0398L Glutathione Resin Genscript Cat# L00206 Casein Kinase II (CKII) NEB Cat# P6010L Guanosine 5 0 -O-(3-thiotriphosphate) (GTPyS) Abcam Cat# ab146662 FITC-labeled Ric-8A (504-530) peptide Analytical Core Facility, Tufts University Custom Synthesis Hygromycin B Invitrogen 10687010 DMEM Invitrogen 11995065 FBS, USDA Invitrogen 10437036 Mixed cellulose membrane EMD Millipore Cat# GSWP02500 HiPrep Phenyl HP 16/10 GE Healthcare Cat# 29018184 SYPRO Orange Life Technologies Corporation Cat# S6650 Deposited Data Ric-8A-Gaq coordinates Ric-8A-Gai1 coordinates Ric-8A-Gaq EM map Ric-8A-Gai1 EM map Experimental Models: Cell Lines This paper PDB: 6VU5 This paper PDB: 6VU7 This paper EMDB: EMD-21387 This paper EMDB: EMD-21388 Trichuplusia ni Expression Systems Cat# 94-002S HEK293T ATCC CRL-11268 HEK293T RIC-8A knockout Papasergi-Scott et al.... The soluble fraction was incubated with Glutathione resin (Genscript) for 1 hour at 4 C. | Get A Quote |
Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A cor... More
