Rational engineering of ratiometric calcium sensors with bright green and red fluorescent proteins

摘要

a Schematic representation of the sensor construct linking the RFP (mScarlet), calcium sensing domain (TnC), and circularly permuted GFP-analog in series. The two inset images represent the fluorescence intensity in the two imaged channels from one example cell. The illumination intensity was 5 mW/mm2. The scale bar is 20 µm. b A schematic illustration shows various sensor designs that maximized on the Ca2+ sensitivity after improvements through amino acid truncation, circularly permutation, sensing domain optimization, linker optimization, and donor optimization. The XFP referred to a set of fluorescent proteins including mNeonGreen, mCitrine, EGFP, mClover3, or mVenus. c The FRET efficiency of the sensor and the quantum yield of the acceptor remained high for truncations of mScarlet shorter than 9 amino acids. Larger truncations not only extinguished the red fluorescence, but also significantly reduced the FRET efficiency between the green and red fluorescent proteins. The horizontal dash represents the reported quantum yield of native mScarlet. (d) Circular permutation of the mCitrine donor at specific locations optimized the FRET efficiency change between the sensors’ Ca2+-resting state and Ca2+-saturated state. e Sensors using the TnC2 calcium sensing domain had significantly higher Ca2+-saturated FRET efficiency (yellow) and the Ca2+-resting state FRET efficiency (green) than those of sensors using the TnC3 calcium sensing domain. f The relationship between the resting-Ca2+ FRET efficiency of sensors and the change in FRET efficiency for various linkers between sensing domains and fluorescent proteins. The optimal linker for each donor (open triangular and circular symbols) had both high resting FRET efficiency and high FRET efficiency change. g The relationship between the various FP pairs’ resting FRET efficiency and change in FRET efficiency between Ca2+-resting and Ca2+-saturated states. Dots represent FRET metrics measured from individual cells, while the symbols represent the mean FRET metrics of each variant. All error bars are s.e.m., n ≥ 10 cells for each sensor. All statistical tests were the two-sided Wilcoxon rank-sum test, where * signifies p < 0.1, ** signifies p < 0.01, *** signifies p < 0.001, n.s. signifies not significant.