American Chemical Society
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Protein–Peptide Binding Energetics under Crowded Conditions

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journal contribution
posted on 2020-10-12, 17:51 authored by Samantha S. Stadmiller, Jhoan S. Aguilar, Stuart Parnham, Gary J. Pielak
Nearly all biological processes, including strictly regulated protein–protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen-activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19F NMR lineshape analysis. We also analyze the temperature dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3–peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically relevant cosolutes. Although most of the protein cosolutes destabilize the SH3–peptide complexes, the effects are nongeneralizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein–protein interactions in cells and show the importance of using physiologically relevant cosolutes for investigating macromolecular crowding effects.