Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided and Genome Mining Strategies

Abstract

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1 (K + ratiometric indicator for optical imaging based on mNeonGreen 1), based on the insertion of a potassium binding protein, Kbp, from E. coli (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured K d of 69 ± 10 mM (mean ± standard deviation used throughout). We identified Ec-Kbp’s binding site using NMR spectroscopy to detect protein–thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION1/D9N and KRaION2, which exhibit isotonically measured K d ’s of 138 ± 21 and 96 ± 9 mM. We identified four Ec-Kbp homologues as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39–112 mM range. KRaIONs functioned in HeLa cells, but the K d values differed from the isotonically measured case. We found that, by tuning the experimental conditions, K d values could be obtained that were consistent in vitro and in vivo . We thus recommend characterizing potassium indicator K d in the physiological context of interest before application.

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…Although the use of main chain binding ligands limits the options for modifying the K + -binding affinities, the re-refined Kbp NMR structure was used to inform the development and optimization of a series of K + ratiometric indicators for optical imaging based on mNeon-Green (KRaION), which have an expanded range of affinities. 77 Here, Kbp was first inserted into the mNeonGreen sequence to generate KRaION1 with a K d of 69 mM and a dynamic range up to 4.3-fold. By introducing rational mutations around the K + -binding site without directly changing the binding residues, the K d range was expanded up to 138 mM (KRaION1/D9N).…”

“…Also, given that the metal-sensing mechanism often depends on a metal ion-induced shift in the chromophore p K a , sensitivity to pH must be checked in each biological system of interest . In some cases, single fluorescent protein sensors have detectable metal ion-induced shifts in excitation or emission spectra and can therefore be used as ratiometric sensors by collecting fluorescence at two different excitation or emission wavelengths. ,− Another approach is to fuse a single fluorescent protein sensor with another fluorescent protein that can be used as an internal standard (Figure D). ,,− …”

“…It is not as selective as the FRET sensors for K + and can respond to high concentrations of Na + ( K d = 153 mM), but it was successfully applied to detect K + in dual-color imaging experiments in dissociated neurons with the Ca 2+ sensor K-GECO1. Improved single fluorescent protein-based K + sensors were developed using structure-guided design. , Although it was initially inferred by the location of the K + -induced conformational changes that K + binding occurred in the BON domain of Kbp, the binding ligands were not identified until the original K + -saturated nuclear magnetic resonance (NMR) structure was re-refined using protein-thallium scalar couplings to indicate the K + -binding site . The K + -binding site utilizes all backbone carbonyl units as the binding ligands and no side chains.…”

“…77,78 Although it was initially inferred by the location of the K + -induced conformational changes that K + binding occurred in the BON domain of Kbp, the binding ligands were not identified until the original K + -saturated nuclear magnetic resonance (NMR) structure was re-refined using protein-thallium scalar couplings to indicate the K + -binding site. 77 The K + -binding site utilizes all backbone carbonyl units as the binding ligands and no side chains. Around the same time, a crystal structure of GINKO1 also elucidated this binding site for Kbp.…”

“…The folding of the protein main chain to bind K + is thought to play a role in the selectivity against the smaller Na + cation, for which the steric constraints would be higher. Although the use of main chain binding ligands limits the options for modifying the K + -binding affinities, the re-refined Kbp NMR structure was used to inform the development and optimization of a series of K + ratiometric indicators for optical imaging based on mNeonGreen (KRaION), which have an expanded range of affinities . Here, Kbp was first inserted into the mNeonGreen sequence to generate KRaION1 with a K d of 69 mM and a dynamic range up to 4.3-fold.…”