Biology subjects

Dodani, S. C.

Publications and source records attributed to Dodani, S. C..

3 recordsLinked to original sources

bioRxiv2022

The ChlorON Series: Turn-on fluorescent protein sensors for imaging labile chloride in living cells

Beyond its role as the queen electrolyte, chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of mNeonGreen (mNG) derived from lanYFP found in the cephalochordate Branchiostoma lanceolatum. Targeted engineering of two non-coordinating residues in the chloride binding pocket of mNG unlocks the ChlorON series. In vitro spectroscopy reveals that the binding of chloride tunes the chromophore environment to give rise to the turn-on response. We further showcase how this unique sensing mechanism can be exploited for directly imaging labile chloride in living cells with spatial and temporal resolution, accelerating the path forward for fundamental and translational aspects of chloride biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/503807v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@36cc4org.highwire.dtl.DTLVardef@d9dccborg.highwire.dtl.DTLVardef@12ec0fforg.highwire.dtl.DTLVardef@80c9db_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↗

bioRxiv2022

Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Our understanding of chloride in biology can be accelerated through the application of fluorescent protein-based sensors in living cells. Laboratory-guided evolution can be used to diversify and identify sensors with specific properties. Recently, we established that the fluorescent proton-pumping rhodopsin wtGR from Gloeobacter violaceus can be converted into a fluorescent sensor for chloride. To unlock this non-natural function, a single point mutation at the Schiff counterion position from D121V was introduced into wtGR fused with cyan fluorescent protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary analysis with directed evolution to understand how the rhodopsin sequence space can be explored and engineered to improve this starting point. We first show how evolutionary couplings are predictive of functional sites in the rhodopsin family and how a fitness metric based on sequence can be used to quantify known proton-pumping activities of GR-CFP variants. Then, we couple this ability to predict potential functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of five residues along the proton-pumping pathway in GR1-CFP. This iterative selection process results in GR2-CFP with four additional mutations, E132K, A84K, T125C, and V245I. Finally, bulk, and single fluorescence measurements in live E. coli reveal that GR2-CFP is a reversible, ratiometric fluorescent sensor for extracellular chloride with an improved dynamic range. We anticipate that our framework will be applicable to other systems, providing a more efficient methodology to engineer fluorescent-protein based sensors with desired properties. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/474067v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1ddb801org.highwire.dtl.DTLVardef@15b4a9corg.highwire.dtl.DTLVardef@7f497corg.highwire.dtl.DTLVardef@b19c5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗