Franchini, L. R. | Search bioRxiv

bioRxiv2026

Phosphorylation tunes electrostatically driven protein-RNA interactions

Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Forster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein-RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

biophysics↗

bioRxiv2024

Human Cells for Human Proteins: Isotope Labeling in Mammalian Cells for Functional NMR Studies of Disease-Relevant Proteins

In biological and biomedical research the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods: it allows studying structure, function and dynamics of biomolecules and, importantly, their interactions with natural ligands or drugs. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in e.g. HEK293 cells are often established, isotope labeling is difficult and very expensive. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells. We demonstrate uniform 15N and 13C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV 13C-methyl labeling pattern. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. This enables NMR studies of important, but difficult to produce proteins, like receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.

biophysics↗