title: Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement.
abstract: Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used 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 fluorescence and 1.3-fold increase in calcium affinity 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 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Chen
authors.forename: Xianqiang
authors.initials: X
authors.lastname: Song
authors.forename: Sheng
authors.initials: S
authors.lastname: Ye
authors.forename: Lin
authors.initials: L
authors.lastname: Miao
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhu
authors.forename: Rong-Guang
authors.initials: RG
authors.lastname: Zhang
authors.forename: Guangju
authors.initials: G
authors.lastname: Ji
mesh_terms: D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D002147:Calmodulin; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium
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