Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions

Abstract

Studies of protein-protein interactions, carried out in polymer solutions, are designed to mimic the crowded environment inside living cells. It was shown that crowding enhances oligomerization and polymerization of macromolecules. Conversely, we have shown that crowding has only a small effect on the rate of association of protein complexes. Here, we investigated the equilibrium effects of crowding on protein heterodimerization of TEM1-beta-lactamase with beta-lactamase inhibitor protein (BLIP) and barnase with barstar. We also contrasted these with the effect of crowding on the weak binding pair CyPet-YPet. We measured the association and dissociation rates as well as the affinities and thermodynamic parameters of these interactions in polyethylene glycol and dextran solutions. For TEM1-BLIP and for barnase-barstar, only a minor reduction in association rate constants compared to that expected based on solution viscosity was found. Dissociation rate constants showed similar levels of reduction. Overall, this resulted in a binding affinity that is quite similar to that in aqueous solutions. On the other hand, for the CyPet-YPet pair, aggregation, and not enhanced dimerization, was detected in polyethylene glycol solutions. The results suggest that typical crowding agents have only a small effect on specific protein-protein dimerization reactions. Although crowding in the cell results from proteins and other macromolecules, one may still speculate that binding in vivo is not very different from that measured in dilute solutions.

Protein–protein interactions in a crowded environment

“…Phillip et al (2009) found a negligible impact on the binding affinity of TEM1-β-lactamase with its inhibitor β-lactamase and barnase with barstar due to increased PEG 1000 crowding that varied up to 30 % packing fraction. Results from experimental studies (Crowley et al 2008;Phillip et al 2009) suggest the presence of an attractive interaction between PEG molecules and proteins. Based on our earlier discussion, attractive protein-crowder interactions will actually counteract the stabilizing effect of excluded volume on complex formation and can help explain this trend.…”

“…Thus, to answer the above questions, many experimental studies have been performed using synthetic polymers or specific proteins as crowding agents. These studies have addressed both the thermodynamics and kinetics of protein-protein interactions in a crowded environment (Minton and Wilf 1981;Minton 1983;Jarvis and Ring 1990;van den Berg et al 1999;Wenner and Bloomfield 1999;Morar et al 2001;Patel et al 2002;Kozer and Schreiber 2004;Zorrilla et al 2004;Phillip et al 2009;Wang et al 2011;Fodeke and Minton 2011). Using "inert" crowding agents, the primary focus in most of these studies was to understand the excluded volume effects of crowding agents on the formation of protein complexes (Minton 1983;Kim et al 2010).…”

“…As expected based on simple theoretical models, these experiments have shown that the consequence of excluded volume effects (entropic in nature) is to force proteins to form a stable complex, thereby increasing the volume available to the crowding agents. However, a few experiments have shown an unexpected trend, i.e., destabilization of protein complexes in the presence of crowding agents (Phillip et al 2009;Jiao et al 2010). This observation can be explained by attractive interactions between proteins and crowding agents, which can inevitably arise from a combination of electrostatic interactions, hydrogen bonding, hydrophobic interactions, and van der Waals interactions.…”

Protein-binding dynamics imaged in a living cell

“…50 kDa), translational diffusion rates are estimated to be reduced by a factor of approximately 5 (17,31,32). Although competitive interactions and slower diffusion may act to reduce binding rates, the depletion force (or the caging effect) acts to enhance it (33)(34)(35). It seems that these factors operate in opposite directions, yielding only a minor modulation of binding rates compared to simple buffer solutions.…”

“…Association rate constants were determined using a stopped-flow fluorescence spectrometer (Applied PhotoPhysics) under pseudo-first-order conditions (20). Dissociation rate constants for both TEM1-BLIP and CyTEM-YBLIP wild-type complexes were determined using the ProteOn XPR36 (BioRad) as described before (34). Dissociation rate constant of the TEM1 E104A -YBLIP WT complex was determined as described in Cell Extract Measurements.…”