Hue-shifted monomeric variants of Clavulariacyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

Abstract

Background: In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.

Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein

“…rxRFP1.4 localized in the ER, as confirmed by colocalization studies with a previously established green fluorescent ER-mWasabi probe [19], responded to treatments of AT-2 (1 mM) and DTT (10 mM), demonstrating its usefulness as a fluorescent probe for redox dynamics in the ER (Figure 4A & 4B). We observed that the direct addition of DTT (10 mM) in the imaging medium was unable to fully reduce rxRFP1.4 in the ER, so we further developed a procedure to permeabilize cell membranes with digitonin.…”

Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

“…In each round the brightest colonies were picked, plasmids isolated, and the pool of improved FP genes used as the template for the subsequent library. We have successfully applied this basic strategy to the optimization of a teal FP (27), blue FPs (17), and a YFP with violet excitation (37).…”

Reporting neural activity with genetically encoded calcium indicators

“…Brighter cyan FPs have recently been developed, such as Cerulean (Rizzo et al, 2004) or mTFP (Ai et al, 2008). Substitution of Cerulean for ECFP in cyan-to-yellow FRET sensors has improved SNR (Heim et al, 2007), though this may decrease photostability (Shaner et al, 2005).…”