Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain.

Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases.

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro.

In vitro Reconstitution of a Membrane Switch Mechanism for the Polarity Protein LGL.

Cell polarity arises from a combination of interactions between biological molecules, such as activation, inhibition, and positive or negative feedback between specific polarity units. Activation and inhibition often take place in the form of a membrane binding switch. Lethal giant larvae (LGL), a conserved regulator of cell polarity in animals, was suggested to function as such a switch.

Diffusion coefficients and dissociation constants of enhanced green fluorescent protein binding to free standing membranes.

Recently, a new and versatile assay to determine the partitioning coefficient [Formula: see text] as a measure for the affinity of peripheral membrane proteins for lipid bilayers was presented in the research article entitled, "Introducing a fluorescence-based standard to quantify protein partitioning into membranes" [1]. Here, the well-characterized binding of hexahistidine-tag (His6) to NTA(Ni) was utilized. Complementarily, this data article reports the average diffusion coefficient [Formula: see text] of His6-tagged enhanced green fluorescent protein (eGFP-His6) and the fluorescent lipid analog ATTO-647N-DOPE in giant unilamellar vesicles (GUVs) containing different amounts of NTA(Ni) lipids.

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites.

Asymmetric supported lipid bilayer formation via methyl-β-cyclodextrin mediated lipid exchange: influence of asymmetry on lipid dynamics and phase behavior.

Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca.