atomic-scale-structural-studies-macromolecular-assemblies-solid-state

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

UMR5248 CNRS, Université de Bordeaux

Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free "precipitate mode" and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.

Cell-Free Expression for the Study of Hydrophobic Proteins: The Example of Yeast ATP-Synthase Subunits.

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide binding sites in RhoGAP domains have been reported to correspond to polybasic regions (PBR), which are located at the unstructured flexible termini of proteins. Solid state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.

James Tolchard

Institute of Chemistry,
Biology of Membranes,
Nanoobjects,
Institute of Chemistry, Biology of Membranes, Nanoobjects

UMR5248 CNRS, Université de Bordeaux

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

James Tolchard

Institute of Chemistry,Biology of Membranes,Nanoobjects,Institute of Chemistry, Biology of Membranes, Nanoobjects

UMR5248 CNRS, Université de Bordeaux

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Institute of Chemistry,
Biology of Membranes,
Nanoobjects,
Institute of Chemistry, Biology of Membranes, Nanoobjects