Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Protein Cell. 2013 Apr;4(4):299-309. doi: 10.1007/s13238-013-2103-4. Epub 2013 Apr 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / chemistry
  • Calcium / metabolism
  • Calmodulin / chemistry
  • Calmodulin / genetics
  • Calmodulin / metabolism
  • Crystallography, X-Ray
  • Dimerization
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Histidine / chemistry
  • Histidine / genetics
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Myosin-Light-Chain Kinase / chemistry
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics

Substances

  • Calmodulin
  • M13 protein (myosin light-chain kinase)
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • polyhistidine
  • Histidine
  • Myosin-Light-Chain Kinase
  • Calcium