Structural Basis for Calcium Sensing by GCaMP2

Structure. 2008 Dec 10;16(12):1817-27. doi: 10.1016/j.str.2008.10.008.

Abstract

Genetically encoded Ca(2+) indicators are important tools that enable the measurement of Ca(2+) dynamics in a physiologically relevant context. GCaMP2, one of the most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca(2+) fluxes in vivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveal a sophisticated molecular mechanism for Ca(2+) sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate that the Ca(2+)-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca(2+)-sensitive probes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium Signaling*
  • Calmodulin / chemistry*
  • Calmodulin / genetics
  • Calmodulin / isolation & purification
  • Calmodulin / metabolism*
  • Crystallization
  • Crystallography, X-Ray
  • Fluorescent Dyes / isolation & purification
  • Fluorescent Dyes / metabolism
  • Green Fluorescent Proteins / isolation & purification
  • Green Fluorescent Proteins / metabolism
  • Hydrogen Bonding
  • Kinetics
  • Light
  • Models, Molecular
  • Myosin Light Chains / chemistry
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Scattering, Small Angle
  • Spectrometry, Fluorescence
  • Ultracentrifugation
  • Water / chemistry
  • X-Ray Diffraction

Substances

  • Calmodulin
  • Fluorescent Dyes
  • Myosin Light Chains
  • Recombinant Fusion Proteins
  • Water
  • Green Fluorescent Proteins