Biology subjects

Tutol, J.

Publications and source records attributed to Tutol, J..

3 recordsLinked to original sources

bioRxiv2026

An All-Optical Approach to Probe Chloride Transport with a Bright ChlorON

Chloride transport across cellular membranes is fundamental to physiology. Yet, this dynamic process remains difficult to capture with existing methods that rely on electrophysiology or indirect iodide-quenching assays, leaving real-time imaging of chloride transport a largely unexplored frontier. To address this gap, we upgrade our first-generation fluorescent protein indicator ChlorON-1 into ChlorON-1-PRO through targeted mutagenesis of an evolutionarily conserved gatepost residue. A single mutation (C139N) preserves the turn-on sensing mechanism (13.9-fold response) while boosting affinity (Kd = 47.4 mM) and bound-state brightness (13.6). Molecular dynamics simulations provide atomic-level insights for these enhancements, supporting a model in which the mutation globally rigidifies the {beta}-barrel and locally prearranges the binding pocket while stabilizing the chromophore. Finally, we showcase the utility of ChlorON-1-PRO for real-time monitoring of endogenous chloride transport under basal and pharmacologically modulated conditions in the U-2 OS cell model.

biochemistry↗

bioRxiv2025

NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells

The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also a protective nutrient and signaling ion - particularly as connected to reactive nitrogen oxides. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from Staphylococcus carnosus. Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (Kd {approx} 9 {micro}M). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4{degrees}. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the {beta}7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/644677v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ba853eorg.highwire.dtl.DTLVardef@81bed9org.highwire.dtl.DTLVardef@18a313org.highwire.dtl.DTLVardef@17a5e80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Directed Evolution of a Genetically Encoded Indicator for Chloride

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/624492v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@8a9309org.highwire.dtl.DTLVardef@1bb6770org.highwire.dtl.DTLVardef@840dd4org.highwire.dtl.DTLVardef@1a7e2cb_HPS_FORMAT_FIGEXP M_FIG C_FIG Inarguably, the green fluorescent protein (GFP) family is an exemplary model for protein engineering, accessing a range of unparalleled functions and utility in biology. The first variant to recognize and provide an optical output of chloride in living cells was serendipitously uncovered more than 25 years ago. Since then, researchers have actively expanded the potential of GFP indicators for chloride through site-directed and combinatorial site-saturation mutagenesis, along with chimeragenesis. However, to date, the power of directed evolution has yet to be unleashed. As a proof-of-concept, here, we use random mutagenesis paired with anion walking to engineer a chloride-insensitive fluorescent protein named OFPxm into a functional indicator named ChlorOFF. The sampled mutational landscape unveils an evolutionary convergent solution at one position in the anion binding pocket and nine other mutations across eight positions, of which only one has been previously linked to chloride sensing potential in the GFP family.

biochemistry↗