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- Proteinstrukturanalyse (1) (remove)
Nanotemplates for the combined structural and functional analysis of membrane-associated proteins
(2019)
Plasma membranes are essential for life because they give cells an identity. Plasma membranes are almost impermeable to fluids and substances. Still, transport between inside and outside needs to be possible. An important transport way is endocytosis. This mechanism relies on membrane-associated proteins that sense and induce curvature to the plasma membrane. However, the physics and structural dynamics behind proteins acting on membranes is not well understood. There is a standard method in vitro to investigate membrane-associated proteins sensing spherical geometries: They are incubated on unilamellar vesicles. This procedure allows to analyze these proteins in their bound state. This approach is inappropriate for GRAF1 (GTPase Regulator Associated with Focal Adhesion Kinase-1), a key player in endocytosis because it senses tubular geometries instead. However, GRAF1 extrudes lipid tubes from vesicles that can be analyzed. Still, this is a limited method because these tubes suffer from inhomogeneity and they do not enable the observation of intermediate and lower concentration binding states. To overcome this issue they can be incubated on pre-tubular structures called nanotemplates. There have been studies using carbon nanotubes and Galactosylceramide lipid tubes as nanotemplates. These approaches require complex chemical modifications or expensive components and they are not necessarily flexible. In this work we present a simple and easy new approach to prepare nanotemplates using Folch lipid mixture. We show on the basis of BPG, a truncate of GRAF1, that our nanotemplates are suitable for Cryo-EM and that it is possible to use IHRSR (Iterative Helical Real Space Reconstruction) to analyze the structure of BPG in its bound state. Moreover, the qualification for Cryo-EM allows to use plunge freezing to interrupt the incubation on our nanotemplates abruptly. This enables the analysis of intermediate binding states to understand the binding process.