Single-molecule FRET and tracking of transfected biomolecules: multi-dimensional protein dynamics in living cells

Proteins in cells exhibit conformational dynamics, equally influenced by dynamic interactions with other biomolecules and their spatial variations, which can be induced by the protein’s compartment. Altogether this multi-dimensional dynamic is difficult to measure in cellula , because of limitations in instrumentation, fluorescence methodologies and the difficulty to track freely diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation (ALEX) based three-channel (donor, acceptor and FRET intensity) tracking with a live-cell HILO microscope. Unexpectedly, we find that the heat shock protein Hsp90 shows different conformational populations in vitro and in cellula . Moreover, Hsp90’s conformational states depend on the localization within the cell, which is demonstrated by comparing a physical (microinjection) and a biological (SLO) transfection method. FRET-TTB ( Tracking of Transfected Biomolecules ) opens the path to study protein conformational dynamics of transfected and native biomolecules in cellula , including time-resolved cellular localization.