Biology subjects

Gatin-Fraudet, B.

Publications and source records attributed to Gatin-Fraudet, B..

3 recordsLinked to original sources

bioRxiv2026

Emergence of new function through evolutionary divergence of an intrinsically disordered region

Intrinsically disordered regions (IDRs) are major drivers of protein functional diversification, yet the molecular features that enable the emergence of new functions within disordered sequences remain poorly understood. FCHO1 and FCHO2 are paralogous pioneer proteins of clathrin-mediated endocytosis that share a conserved domain architecture but perform distinct cellular functions and cannot compensate for each other's loss. Here we show that functional divergence between these proteins is associated with the acquisition of transient structure within their disordered regions. Using nuclear magnetic resonance spectroscopy, we identify two highly populated -helical elements in the IDR of FCHO1 that are absent from FCHO2. One of these helices mediates FCHO1 self-association and drives intracellular assembly, whereas FCHO2 lacks this behavior. Introduction of the FCHO1 helix into FCHO2 is sufficient to confer self-association and cellular assembly, demonstrating that a transient structural element embedded within an IDR can act as a transferable functional module. Evolutionary analysis reveals that this helical propensity emerged following duplication of the ancestral FCHO gene and became progressively reinforced during evolution. Despite this divergence, the same region retains a conserved membrane-binding activity in both paralogs. Together, our findings show how acquisition of transient secondary structure within an intrinsically disordered region can generate new molecular behaviors while preserving ancestral functions, providing a mechanism for the functional specialization of paralogous proteins.

biophysics↗

bioRxiv2026

HaloTag Ligand and HaloTag Protein engineering for a binary fluorescent turn-on probe

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

synthetic biology↗

bioRxiv2025

A silicon rhodamine-fused glibenclamide to label and detect malaria-infected red blood cells

The malaria parasite Plasmodium falciparum affects the lives of millions of people worldwide every year. The detection of replicating parasites within human red blood cells is of paramount importance, requiring appropriate diagnostic tools. Herein, we design and apply a silicon rhodamine-fused glibenclamide (SiR-glib). We first test this far-red fluorescent, fluorogenic and endoplasmic reticulum-targeting sulfonylurea in mammalian cells and pancreatic tissues, before characterizing its labeling performance in red blood cells infected with the asexual developmental stages of Plasmodium falciparum. We further combine SiR-glib with a portable smartphone-based microscope to easily and rapidly identify parasitized red blood cells, providing proof of principle for diagnostic use in rural endemic areas without major healthcare facilities.

biochemistry↗