Green Fluorescent Proteins

We use Green fluorescent Protein (GFP) and its colour variants (CFP, YFP etc) to investigate conformational changes in myosin using Fluorescence Resonance Energy Tranfser (FRET) and also as probes in single molecule assays. Single molecule fluorescence measurements of Yellow Fluorescent Protein (YFP) show that the emission blinks on the seconds time scale owing to a slow proton-coupled isomerisation step. In addition, prolonged exposure to 514 nm light leads to a reversibly-bleached state that can be reactivated by flashing with 350 nm light, but regeneration of the fluorophore (i.e. YFP- anion) is limited by the reverse of the proton-coupled isomerisation step. These findings have implications for YFP applications in cell biology, including use as a FRET acceptor or donor.

 

GFP

Single GFP - myosin motors attached to actin filaments imaged using TIRF microscopy

See Alberts et al (2007) Molecular Biology of the Cell (5th edition Fig 9-36) for an introduction to TIRF microscopy based on this figure.

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Department of Molecular and Cell Biology

T: +44(0)116 229 7038
E: MolCellBiol@le.ac.uk

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