Uorescent Atto488linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras

Uorescent 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 in the loop between beta strand 3 (3) and alpha helix 2 (2) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either aspect of, or allosterically coupled to, the dimer interface. The 2D dimerization Kd is measured to be around the order of 1 103 molecules/m2, inside the broad range of Ras surface densities measured in vivo (10, 335). Dimerization only happens on the membrane surface; H-Ras is strictly monomeric at comparable densities in remedy, suggesting that a membrane-inducedstructural change in H-Ras results in dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive for the specifics of HVR lipidation, suggesting that dimerization is a general property of H-Ras on membrane surfaces. ResultsH-Ras Exhibits Decreased Translational and Rotational Mobility on Supported Membranes. In these experiments, Ras(C181) or Ras(C181,C184)are attached to the membrane through coupling of cysteines C181 and C184 in the HVR to NMDA Receptor Agonist review maleimide functionalized lipid, 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-DOPE) (Fig. 1A). Since MCCDOPE is totally miscible within the lipid bilayer, clustering because of the lipid anchor itself is avoided. In native H-Ras, palmitoylation takes location within the identical two cysteine residues, C181 and C184. Two-color FCS permits the translational mobility of lipids and membrane-linked H-Ras to be monitored simultaneously from the very same spot (Fig. 1B). A modest percentage (0.005 mol ) of Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) lipid is included within the membrane, whereas H-Ras is loaded with fluorescent nucleotide, Atto488-GDP or Atto488 ppNp. Normalized autocorrelation functions, G(), of fluorescence fluctuations inside the lipid and Ras(C181) channels are illustrated in Fig. 1C. Measured autocorrelation instances correspond to diffusion coefficients, D, of three.39 0.15 m2/s and 1.12 0.04 m2/s for TRDHPE lipid and Ras(C181) respectively. Ras(C181) exhibits quicker mobility than the doubly anchored Ras(C181,C184) constructs, supplying confirmation that both anchor websites are coupled to lipids.Fig. 1. Lateral diffusion of H-Ras on membranes. (A) Two feasible H-Ras orientations when tethered onto a lipid membrane (modified from ref. 18). The secondary structure of H-Ras G-domain (aa 166) is shown in cartoon mode. The portion of HVR (aa 16784) used in the present function is in orange just above the prime leaflet of the bilayer (gray). The lipid anchor, MC4R Agonist medchemexpress MCC-DOPE, is not integrated. (B) Schematic of two-color FCS setup. (C) Normalized auto-correlation functions, G(), of Ras(C181)-GDP and TR lipid at an H-Ras surface density of 312 molecules/m2. The diffusion time constants, trans, are normalized to the detection area. The calculated diffusion coefficients are 3.39 0.15 m2/s and 1.12 0.04 m2/s for lipid and H-Ras, respectively. (D) G() of Ras(Y64A,C181)GDP and TR lipid at a Ras(Y64A,C181) surface density of 293 molecules/m2 having a calculated D of 3.39 0.05 m2/s and three.16 0.07 m2/s, respectively. (E) Diffusion step-size h.