A high-resolution crystal structure of the genetically-encoded calcium indicator G-CaMP2 would aid in rational design of improved calcium indicators. Crystallization of G-CaMP2 was first reported here :
Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2
Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.
High-resolution atomic structures and mutational analysis were presented at SfN 2008 (see this previous post)
However, today a competing group has published an independent report on a similar set of G-CaMP2 structures in Cell Structure. More details to come…
Genetically encoded Ca2+ indicators are important tools that enable the measurement of Ca2+ dynamics in a physiologically relevant context. GCaMP2, one ofthe most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca2+ fluxes invivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveala sophisticated molecular mechanism for Ca2+ 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 thatthe Ca2+-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 Ca2+-sensitive probes.