ABSTRACT
Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.
Subject(s)
Biotechnology/methods , Luminescent Proteins , Whole Body Imaging/methods , Amino Acid Sequence , Animals , Biotechnology/instrumentation , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , HeLa Cells , Humans , Infrared Rays , Luminescent Proteins/genetics , Luminescent Proteins/toxicity , Mice , Molecular Sequence Data , Protein Multimerization , Protein Stability , Sequence Alignment , Transfection , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca(2+) sensor, Case16, in the presence of a low Ca(2+) concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca(2+)-dependent response when only two out of four Ca(2+)-binding pockets of calmodulin (CaM) are occupied with Ca(2+) ions. In such a "half Ca(2+)-bound state", Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca(2+) sensor Case12 at saturating Ca(2+) concentration. Based on this structure, we postulate that cpGFP-based Ca(2+) sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca(2+) sensors in living cells.
