K-Ras4B Remains Monomeric on Membranes over a Wide Range of Surface Densities and Lipid Compositions

Abstract

Ras is a membrane-anchored signaling protein that serves as a hub for many signaling pathways and also plays a prominent role in cancer. The intrinsic behavior of Ras on the membrane has captivated the biophysics community in recent years, especially the possibility that it may form dimers. In this article, we describe results from a comprehensive series of experiments using fluorescence correlation spectroscopy and single-molecule tracking to probe the possible dimerization of natively expressed and fully processed K-Ras4B in supported lipid bilayer membranes. Key to these studies is the fact that K-Ras4B has its native membrane anchor, including both the farnesylation and methylation of the terminal cysteine, enabling detailed exploration of possible effects of cholesterol and lipid composition on K-Ras4B membrane organization. The results from all conditions studied indicate that full-length K-Ras4B lacks intrinsic dimerization capability. This suggests that any lateral organization of Ras in living cell membranes likely stems from interactions with other factors.

Figure 1

Experimental design for K-Ras on SLBs. (A) Given here are processed K-Ras4B constructs used in these experiments: wild-type (top) and eGFP-labeled (bottom). (B) Cross-linked dimers formed by RBD fused to LeuZ were used as benchmarks for dimerization for the diffusion measurements. (C) Given here is the supported lipid bilayer-based experimental setup for FCS (top), and resulting autocorrelation traces for monomeric and cross-linked Ras (bottom). (D) Shown here is the TIRF microscopy setup for SMT (top), and step-size distribution (bottom). (Inset) This shows an example of SMT trajectories from which the step-size distributions are obtained; scale bar: 10 μm. To see this figure in color, go online.

Figure 2

Theoretical dynamic range for FCS in dimerization detection. (A) Fraction of dimers as a function of surface density for a series of 2D dissociation constants K_d are shown. (B) Calculated surface density-titration experiment from conditions given in (A), using experimentally determined diffusion coefficients for monomers and dimers, showing that even weak dimerization up to K_d = 25,000 μm⁻² should produce a detectable change in diffusion.

Figure 3

Density titration diffusion measurement on PS bilayers. (A) FCS measurements of eGFP-K-Ras4B in excess of various guanosine nucleotides were titrated on SLBs containing 20% PS bilayers, with and without 0.5 μM RBD-LeuZ cross linker. (Solid lines) Shown here is the diffusion change if the dimerization occurs with K_d = 125,000 μm⁻², which is essentially a dense-packed concentration. (Dotted lines) Shown here are fits for cross-linked Ras, with K_d values ranging between 300 and 400 μm⁻². (B) (Left) Given here is the SMT step-size distribution for low- and high-density Ras, as well as high-density Ras with RBD-LeuZ. (Right) Given here are density-dependent diffusion coefficients D and fraction in faster species α for Ras without RBD-LeuZ. Both α and D of the two diffusing species are density-invariant, indicating that Ras dimerization does not occur under these circumstances.

Figure 4

Effect of various lipids. (A) Chemical structures of anionic lipids used are shown. DOPS, SOPS, and DOPG have −1 nominal charge, and PIP₂ has −3 nominal charge. SOPS has asymmetric chains (18:0-18:1), whereas the others have symmetric chains (18:1-18:1). (B) FCS measurements of eGFP-K-Ras (left) and wild-type K-Ras (right) on SLBs containing these lipids (charge normalized to −10%) display density-independent diffusion, indicating a monomeric behavior. (C) SMT step size distributions are identical for high- and low-density K-Ras for DOPG or PIP₂ bilayers, regardless of the nucleotide states.

Figure 5

Effect of cholesterol. (A) Intact GUVs were ruptured onto a glass surface to create patches of bilayers containing cholesterol for FCS (B, focus spot shown) and SMT (C, particle trajectories shown) diffusion measurements. Scale bar: 10 μm. (D) Shown here are FCS measurements for (B). (E) Shown here are SMT measurements for (C). K-Ras diffusion is density-independent for all cases.