Biology subjects

Maydew, C.

Publications and source records attributed to Maydew, C..

2 recordsLinked to original sources

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↗

bioRxiv2022

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have been primarily engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the paring of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/499230v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@9c0daaorg.highwire.dtl.DTLVardef@14553cforg.highwire.dtl.DTLVardef@817107org.highwire.dtl.DTLVardef@23a6ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗