The design and evolution of fluorescent protein-based sensors for monoatomic ions in biology

Abstract

Living cells rely on a finely tuned symphony of inorganic ion gradients composed of both cations and anions. This delicate balance is maintained by biological receptors all acting in concert to selectively recognize and position ions for homeostasis. These dynamic processes can be intercepted and visualized with optical microscopy at the organismal, tissue, cellular and subcellular levels using fluorescent protein-based biosensors. Since the first report of such tool for calcium (Ca2+) in 1997, outstanding biological questions and innovations in protein engineering along with associated fields have driven the development of new biosensors for Ca2+ and beyond. In this Review, we summarize a workflow that can be used to generate fluorescent protein-based biosensors to study monoatomic ions in biology. To showcase the scope of this approach, we highlight recent advances reported for Ca2+ biosensors and in detail discuss representative case studies of biosensors reported in the last four years for potassium (K+), magnesium (Mg2+), copper (Cu2+/+), lanthanide (Ln3+) and chloride (Cl−) ions.

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

“…18,19,[21][22][23] Building off this starting point, rational design and mutagenesis strategies have provided access to new sensors with improved properties such as dynamic range, binding affinity, pH dependence, oligomerization state, and/or excitation/emission profiles. 19,22,[24][25][26][27][28][29][30][31][32][33] While these advances have enabled the discovery of new roles for chloride in normal physiology and disease, sensor design has been largely derived from the avGFP scaffold. 24,[34][35][36][37][38] Notably, the red-fluorescent protein EqFP578 from the sea anemone Entacmaea quadricolor has been used as a scaffold to generate mBeRFP-based sensors.…”

The ChlorON Series: Turn-on Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

“…Subsequent studies have applied protein engineering methods around the chromophore and anion binding pocket to further enhance avGFP and avYFP photostability, protein folding, and binding a nity for chloride. 12,[16][17][18][19][20] The engineered E 2 GFP and avYFP-H148Q variants are well-known for elucidating the chloride binding mechanism. From these studies, chloride is known to interact with the T203Y residue, which also forms ⇡ ⇡ stacking interactions with the Y66 chromophore in avYFP-H148Q and E 2 GFP (Figure S1); thus, resulting in a turn-o↵ fluorescence response either through a shift in chromophore pK a or static quenching, respectively.…”

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

“…18,19,21,22 Building off this starting point, rational design and mutagenesis strategies have provided access to new sensors with improved properties such as dynamic range, binding affinity, pH dependence, oligomerization state, and/or excitation/emission profiles. 19,[22][23][24][25][26][27][28][29][30][31][32] While these advances have enabled the discovery of new roles of chloride in normal physiology and disease, sensor design has been largely derived from the avGFP scaffold. 23,[33][34][35][36][37] Notably, the red-fluorescent protein EqFP578 from the sea anemone Entacmaea quadricolor has been used as a scaffold to generate mBeRFP-based sensors.…”