Bimodality in Ras signaling originates from processivity of the Ras activator SOS without deterministic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of deterministic bistability but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts deterministic bistability and may be more resistant to pharmacological inhibition.

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane.

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Native mass spectrometry beyond ammonium acetate: effects of nonvolatile salts on protein stability and structure.

Native mass spectrometry is widely used to probe the structures, stabilities, and stoichiometries of proteins and biomolecular complexes in aqueous solutions, typically containing volatile ammonium acetate or ammonium bicarbonate buffer. In this study, nanoelectrospray emitters with submicron tips are used to produce significantly desalted ions of RNase A and a reduced, alkylated form of this protein, RA-RNase A, from solutions containing 175 mM ammonium acetate, as well as sodium chloride and Tris containing solutions with the same nominal ionic strength and pH. The charge-state distributions formed by nanoelectrospray ionization and tyrosine fluorescence emission data as a function of temperature from these solutions indicate that the folded form of RA-RNase A in solution is stabilized when ammonium acetate is replaced by increasing quantities of NaCl and Tris. Ion mobility data for the 7+ charge state of RA-RNase A indicates that the protein conformation in ammonium acetate changes with increasing concentration of NaCl which stablizes more compact structures. These results are consistent with observations reported 130 years ago by Hofmeister who found that ion identity can affect the stabilities and the structures of proteins in solution. This study indicates the importance of buffer choice when interpreting native mass spectrometry data.