Quantifying Tubulin Heterodimer Assembly in vitro and Cells

As biophysics researchers delve deeper into biomolecular interaction networks, it becomes clear that many weak, nonspecific, transient interactions play a role in living cells. While extensive studies have been made to characterize strong “on-off” protein interactions in vitro, we are particularly interested in studying the role of the cellular environment on protein weak interactions. Here we present a new method to quantify protein-protein weak interactions in living cells. Our model system used human U2-OS cells as a substrate to determine the binding affinity of tubulin-like proteins BtubA and BtubB from the bacterial genus Prosthecobacter. The binding equilibrium was performed with microinjection and monitored using fluorescence resonance energy transfer. Comparing to in vitro results, we found that the cytoplasm matrix promotes the binding affinity of BtubA/B. The promotion is mainly contributed by macromolecular crowding and non-specific sticking effects in cells. A Monte-Carlo-sampling-based model is also developed to simulate the binding promotion from the cytoplasm matrix and fits our experimental results.