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.

Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling.

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron-donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3 orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple centers. New electrophiles and their corresponding alkyne conjugates were profiled directly in cultured cells, achieving thiol saturation in a few minutes at submillimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable the mass spectrometry discovery of >3000 reactive and/or accessible thiols labeled in their native cellular environments in a fraction of the standard analysis time. Surprisingly, only half of the annotated cysteines were identified by both iodoacetamide-desthiobiotin and methylsulfonylbenzothiazole-desthiobiotin in replicate experiments, demonstrating complementary detection by mass spectrometry analysis. These probes offer advantages over existing cysteine alkylation reagents, including accelerated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide analysis and covalent ligand discovery.

Structural Destabilization of Azurin by Imidazolium Chloride Ionic Liquids in Aqueous Solution.

Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain length, and longer-chain ILs can form micelles in aqueous solution. We have investigated the effects of imidazolium chloride ILs on the structure and stability of azurin, which is a very stable Cu2+ redox protein with both α-helix and ß-sheet domains. Temperature-dependent infrared (IR) and vibrational circular dichroism spectroscopy can provide secondary-structure-specific information about how the protein is affected, and temperature-jump transient IR measurements can quantify the IL-influenced unfolding dynamics. Using these techniques, we can quantify how azurin is destabilized by 1.0 M ILs in aqueous solution. The shorter, less hydrophobic ILs, 1-butyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium chloride likely interact with the α-helix domain and decrease protein melting temperature from 82 °C without IL to 55 °C and disturb the overall tertiary structure, resulting in a looser, more open shape. Thermodynamic analysis indicates that protein destabilization is due to increased unfolding entropy. 1-Octyl-3-methylimidazolium chloride [OMIM]Cl, which forms micelles in solution that may partially solvate the protein, has a more significant destabilizing effect, resulting in a melting temperature of 35 °C, larger unfolding entropy, and relaxation kinetics several orders of magnitude faster than with unperturbed azurin. The temperature-independence of the relaxation time constant suggests that in the presence of [OMIM]Cl, the protein folding potential energy surface has become very smooth.

An Experimental and Molecular Dynamics Study of Red Fluorescent Protein mCherry in Novel Aqueous Amino Acid Ionic Liquids.

The search for biocompatible ionic liquids (ILs) with novel biochemical and biomedical applications has recently gained greater attention. In this report, we characterize the effects of two novel amino acid-based aqueous ILs composed of tetramethylguanidinium (TMG) and amino acids on the structure and stability of a widely used red fluorescent protein (mCherry). Our experimental data shows that while the aspartic acid-based IL (TMGAsp) has effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), the alanine-based IL (TMGAla) has a much stronger destabilization effect on the protein structure. Addition of 0.30 M TMGAla to mCherry decreases the unfolding temperature from 83 to 60 °C. Even at 25 °C, TMGAla results in a blue shift of the mCherry absorbance and fluorescence peaks and an increased Stokes shift. Molecular dynamics simulations show that the chromophore conformation and its interaction with mCherry with TMGAla are changed relative to those with TMGAsp or in the absence of ILs. Protein-ILs contact analysis indicates that the mCherry-Asp interactions are hydrophilic but the (fewer) mCherry-Ala interactions are more hydrophobic and may modulate the TMG interaction with the protein. Hence, the anion hydrophobicity may explain the special TMGAla destabilization of mCherry.