Single-fluorophore biosensors based on conformation-sensitive GFP variants.

ABSTRACT: The β-strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in β-strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in β-strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calciu

SUBMITTER: Bonnot A 

PROVIDER: S-EPMC3929668 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature