Protein-binding dynamics imaged in a living cell.
Historically, rate constants were determined in vitro and it was unknown whether they were valid for in vivo biological processes. Here, we bridge this gap by measuring binding dynamics between a pair of proteins in living HeLa cells. Binding of a β-lactamase to its protein inhibitor was initiated by microinjection and monitored by Förster resonance energy transfer. Association rate constants for the wild-type and an electrostatically optimized mutant were only 25% and 50% lower than in vitro values, whereas no change in the rate constant was observed for a slower binding mutant. These changes are much smaller than might be anticipated considering the high macromolecular crowding within the cell. Single-cell analyses of association rate constants and fluorescence recovery after photobleaching reveals a naturally occurring variation in cell density, which is translated to an up to a twofold effect on binding rate constants. The data show that for this model protein interaction the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell.
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Yael
Y
Phillip
Vladimir
V
Kiss
Gideon
G
Schreiber
D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008845:Microinjections; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
D011506:Proteins