Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Gα_i1

Abstract

In order to understand the molecular mechanisms by which G-protein-coupled receptors (GPCRs) activate G proteins, the K349P mutant of G i1 (K349P), which is unable to couple to the muscarinic acetylcholine receptor, was prepared and its crystals were grown along with those of wild-type G i1 protein (WT). The two proteins were crystallized under almost identical conditions, thus enabling a detailed structural comparison. The crystallization conditions performed well irrespective of the identity of the bound nucleotide (GDP or GTPS) and the crystals diffracted to resolutions of 2.2 Å (WTÁGDP), 2.8 Å (WTÁGTPS), 2.6 Å (K349PÁGDP) and 3.2 Å (K349PÁGTPS).

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

“…Crystallization, Data Collection, Structure Determination, and Refinement-Crystals of GDP-and GTP␥S-bound forms of the K345L G␣ i1 variant protein were grown under the same conditions using the hanging-drop vapor diffusion method and previously reported reservoir conditions (37). Briefly, purified protein in 80 mM HEPES, 120 mM succinic acid, 8 mM DTT, pH 8.0, was incubated with either 1 mM GTP␥S or 20 M GDP, 40 M AlCl 3 , and 16 mM NaF.…”

Biochemical characterization of a heterotrimeric Gi-protein activator peptide designed from the junction between the intracellular third loop and sixth transmembrane helix in the m4 muscarinic acetylcholine receptor

“…Glutathione S-transferase (GST)-fused Gai1 (GST-Gai1) was expressed in Escherichia coli BL21 Star (DE3) cells and purified as previously described [16]. The expression construct for the Maltose Binding Protein (MBP)-fused Gai1 (MBP-Gai1) encoded the Gai1 amino acid sequence (residues 30e354) and was expressed in pMAL-c2x (New England Biolabs) using the restriction enzyme recognition sites EcoRI and SalI.…”

Dissociation of Membrane-Anchored Heterotrimeric G-Protein Induced by GαSubunit Binding to GTP

“…Unfortunately, available X-ray data only describe the initial (G αβγ :GDP), , the nucleotide empty complex (G αβγ ), and the final states (G α :GTP + G βγ ) − of this activation mechanism, the possible intermediate conformations/motions of the protein still remaining unsolved. Among other important questions, it can still be debated whether the observed local conformational changes around the GTP ligand are directly due to the GDP:GTP exchange in the G α subunit, or if they could result from dissociation of the whole heterotrimer.…”