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.
Subject(s)
Calcium , Chemistry , Metabolism , Calmodulin , Chemistry , Genetics , Metabolism , Crystallography, X-Ray , Dimerization , Green Fluorescent Proteins , Chemistry , Genetics , Metabolism , Histidine , Chemistry , Genetics , Metabolism , Hydrogen-Ion Concentration , Myosin-Light-Chain Kinase , Chemistry , Genetics , Metabolism , Peptide Fragments , Chemistry , Genetics , Metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins , Chemistry , GeneticsABSTRACT
Objective To investigate the effects of airborne fine particulate matter(PM2.5) on anti-oxidative enzymes activities and lipid peroxidation levels in livers, spleens, and kidneys of rats. Methods 32 male Wistar rats were randomly divided into PM2.5 exposure groups of different concentration (1.5, 7.5, 37.5 mg/kg), exposed by tracheoperfusion and control group treated with physiological saline. Rats were killed 24 h after treatment, and the levels of thiobarbituric acid reactive substance (TBARS), glutathione(GSH) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) were determined. Results PM2.5 exposure caused significantly decrease of SOD, CAT, GSH-Px, SOD/TBARS in livers and kidneys in a dose-dependent manner compared with control group (P