Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23-49 degrees C showed that the cell environment influences protein stability and folding rate. FR... More
Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23-49 degrees C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.
