Effect of Crowding on Protein–Protein Association Rates: Fundamental Differences between Low and High Mass Crowding Agents

Physiological media constitutes a crowded environment that serves as the field of action for protein–protein interaction in vivo. Measuring protein–protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein–protein association and its rate constants (kon) of the β-lactamase (TEM)–β-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0–40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute kon, but not koff values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel kon depends inversely on the solution viscosity. In high mass polymer solutions kon changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp). Polymers that are long enough to form a flexible coil (L/Lp>2) behave as high molecular mass polymers and those who are unable to do so (L/Lp<2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed kon of wt-TEM and three BLIP analogs with up to 100-fold increased values of kon due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment.