Crowding and Protein Dimerization

Abstract

In cells, proteins are surround by macromolecules at concentrations of greater than 100 g/L, yet the majority of our knowledge comes from experiments conducted in dilute buffer solutions. The structure and stability of proteins in cells is influenced by two interactions, hard core repulsions, which arise from excluded volume effects, and transient chemical interactions with surrounding molecules. High concentrations of inert polymers, small molecule cosolutes, and proteins are often used to mimic cellular conditions. Here, we describe the effects of crowding on a protein-protein interaction by using 19F NMR spectroscopy and a variant of the 6 kDa globular B1 domain of protein G. The A34F variant was previously shown to form a side by side dimer in buffer. Using the 3-flourotyorsine labeled variant we measured a dissociation constant of 59 ± 5 μM, consistent with previous the study. We then proceed to show the influences of co-solutes on dimer dissociation. Crowding agents such as sucrose, Ficoll-70, glycine betaine, trimethylamineoxide, and bovine serum albumin stabilize the dimer, whereas urea, ethylene glycol, 8 kDa polyethylene glycol and lysozyme destabilize the dimer. Measuring the temperature dependence of dimerization allows for us to measure the van’t Hoff enthalpy of dimer formation. We are now extending this methodology to in-cell NMR studies of the dimer.