RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

A joint experimental and computational study investigates the translocation of a tryptophan molecule through a phospholipid membrane. Time dependent spectroscopy of the tryptophan side chain determines the rate of permeation into 150 nm phospholipid vesicles. Atomically detailed simulations are conducted to calculate the free energy profiles and the permeation coefficient. Different charging conditions of the peptide (positive, negative, or zwitterion) are considered. Both experiment and simulation reproduce the qualitative trend and suggest that the fastest permeation is when the tryptophan is positively charged. The permeation mechanism, which is revealed by Molecular Dynamics simulations, is of a translocation assisted by a local defect. The influence of long-range electrostatic interactions, such as the membrane dipole potential on the permeation process, is not significant.

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped towards the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ~3 cm−1 greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in a value of 8 – 11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

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RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.