Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantl...Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantly better fluorescent intensity.In this study,we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ.GCaMPJ has a 1.5-fold increase in fl uorescence and 1.3-fold increase in calcium affi nity over GCaMP3.Upon Ca^(2+) binding,GCaMP3 exhibits both monomeric and dimeric forms.The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance.However,GCaMPJ seldom forms dimers under conditions similar to GCaMP3.St ructural and mutagenesis studies on Tyr-380 confi rmed its importance in blocking the cpEGFPβ-barrel holes.Our study proposes an efficient tool for mapping Ca^(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.展开更多
基金supported by grants from the National Basic Research Program of China(Nos.2011CB8091004 and 2009CB918701)。
文摘Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantly better fluorescent intensity.In this study,we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ.GCaMPJ has a 1.5-fold increase in fl uorescence and 1.3-fold increase in calcium affi nity over GCaMP3.Upon Ca^(2+) binding,GCaMP3 exhibits both monomeric and dimeric forms.The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance.However,GCaMPJ seldom forms dimers under conditions similar to GCaMP3.St ructural and mutagenesis studies on Tyr-380 confi rmed its importance in blocking the cpEGFPβ-barrel holes.Our study proposes an efficient tool for mapping Ca^(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
