A membrane (80). As an example, H-Ras localizes to cholesterol-sensitive membrane domains, whereas K-Ras does not (11). A popular C-terminal S-farnesyl moiety operates in concert with one (N-Ras) or two (H-Ras) palmitoyl groups, or having a simple sequence of six lysines in K-Ras4B (12), to provide the major membrane anchorage. Importantly, the G-domain (residues 166) and the hypervariable area (HVR) (residues 16789) dynamically modulate the lipid anchor localization preference to switch in between distinct membrane populations (13). As an example, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is required for efficient activation on the effector Raf and GTP loading of the G-domain promotes this redistribution by a mechanism that demands the HVR (14). Nevertheless, the molecular information of the coupling amongst lipid anchor partitioning and nucleotide-dependent protein embrane interactions stay unclear.W.-C.L. and L.I. contributed equally to this perform. Present address: Division of Chemistry, Nanoscience Center, Bionanotechnology and Nanomedicine Laboratory (BNL), University of Copenhagen, 2100 Copenhagen, Denmark. To whom correspondence should be addressed. E-mail: [email protected] article contains supporting data on the web at www.pnas.org/lookup/suppl/doi:ten. 1073/pnas.1321155111/-/DCSupplemental.2996001 | PNAS | February 25, 2014 | vol.7-Ketolithocholic acid web 111 | no.www.pnas.org/cgi/doi/10.1073/pnas.in vitro (31), but for the reason that artificial dimerization of GST-fused H-Ras leads to Raf activation in resolution, it has been hypothesized that Ras dimers exist on membranes (32). Nevertheless, presumed dimers had been only detected soon after chemical cross-linking (32), along with the intrinsic oligomeric properties of Ras remain unknown. Here, we use a mixture of time-resolved fluorescence spectroscopy and microscopy to characterize H-Ras(C118S, 181) and H-Ras(C118S, 184) [referred to as Ras(C181) and Ras (C181,C184) from right here on] anchored to supported lipid bilayers.3-Aminobutanoic acid manufacturer By tethering H-Ras to membranes at cys181 (or both at cys181 and cys184) by means of a membrane-miscible lipid tail, we get rid of effects of lipid anchor clustering when preserving the HVR area in between the G-domain along with the N-terminal palmitoylation web site at cys181 (or cys184), which is predicted to undergo large conformational changes upon membrane binding and nucleotide exchange (18).PMID:24631563 Labeling is accomplished by way of a fluorescent Atto488linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras types surface density-dependent clusters. Photon counting histogram (PCH) analysis and single-molecule tracking (SMT) reveal that H-Ras clusters are dimers and that no higher-order oligomers are formed. A Y64A point mutation within the loop among beta strand 3 (3) and alpha helix 2 (two) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either a part of, or allosterically coupled to, the dimer interface. The 2D dimerization Kd is measured to become on the order of 1 103 molecules/m2, inside the broad array of Ras surface densities measured in vivo (10, 335). Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in remedy, suggesting that a membrane-inducedstructural modify in H-Ras results in dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive to the specifics of HVR lipidation, suggesting.
