Tinct Glycopeptide Purity & Documentation membrane populations (13). For instance, repartitioning of H-Ras away from cholesterol-sensitive
Tinct membrane populations (13). For instance, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is important for effective activation from the effector Raf and GTP loading with the G-domain promotes this redistribution by a mechanism that requires the HVR (14). On the other hand, the molecular particulars on 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: Department of Chemistry, Nanoscience Center, Bionanotechnology and Nanomedicine Laboratory (BNL), University of Copenhagen, 2100 Copenhagen, Denmark. To whom correspondence really should be addressed. E-mail: jtgroveslbl.gov.This short article contains supporting facts online at pnas.orglookupsuppldoi:10. 1073pnas.1321155111-DCSupplemental.2996001 | PNAS | February 25, 2014 | vol. 111 | no.pnas.orgcgidoi10.1073pnas.in vitro (31), but because artificial dimerization of GST-fused H-Ras leads to Raf activation in resolution, it has been hypothesized that Ras dimers exist on membranes (32). Even so, presumed dimers were only detected after chemical cross-linking (32), and also the intrinsic oligomeric properties of Ras stay 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 here on] anchored to supported lipid bilayers. By tethering H-Ras to membranes at cys181 (or each at cys181 and cys184) by means of a membrane-miscible lipid tail, we get rid of effects of lipid anchor clustering although preserving the HVR region involving the G-domain along with the N-terminal palmitoylation web-site at cys181 (or cys184), which can be predicted to undergo significant conformational adjustments upon membrane binding and nucleotide exchange (18). Labeling is achieved by means of a fluorescent Atto488linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras forms surface density-dependent clusters. Photon counting histogram (PCH) evaluation 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 amongst beta strand three (three) and alpha helix two (2) abolishes dimer formation, suggesting that the corresponding switch II (SII) area is either part of, or allosterically coupled to, the dimer interface. The 2D dimerization Kd is measured to become around the order of 1 103 moleculesm2, inside the broad range of Ras surface densities measured in vivo (ten, 335). Dimerization only happens around the membrane surface; H-Ras is strictly monomeric at comparable densities in solution, suggesting that a membrane-inducedstructural transform in H-Ras results in dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive towards the particulars of HVR lipidation, suggesting that dimerization is usually a general home of H-Ras on membrane surfaces. ResultsH-Ras Exhibits Lowered Translational and Rotational Mobility on Supported Membranes. In these experiments, Ras(C181) or Ras(C181,C184)are attached towards the membrane by means of coupling of cysteines C181 and C184 within the HVR to maleimide functionalized lipid, 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (Akt2 Purity & Documentation MCC-DOPE) (Fig. 1A). Due to the fact MCCDOPE is fully miscible inside the lipid bilayer, clustering because of this.