Hydrophobic Interactions at the Dimer Interface of Glutathione Synthetase

Protein‐protein association involves several types of interactions, including electrostatic, hydrogen bonding, van der Walls, and importantly the hydrophobic effect. Since hydrophobic interactions are responsible for protein folding and stability, they are likely to be important for subunit interactions of multimeric proteins. Some multimeric proteins are cooperative, thus inter‐subunit communication must go through the subunits' interface. Glutathione synthetase (hGS), a negatively cooperative homodimer, has two conserved hydrophobic residues at the interface, Val44 and Val45, and therefore is a good model system. When Val44 and Val45 are mutated to Alanine, the activity of the mutant enzymes are slightly less than wildtype hGS and still maintain negative cooperativity. However, Differential Scanning Calorimetry shows that these mutant enzymes have a decreased transition midpoint (T m ) as compared to wildtype. Our data suggest that the interfacial residues Val44 and Val45, at the interface of human glutathione synthetase, are required to maintain protein stability. (Supported in part by NIH R15GM086833 (MEA), CASCaM (TRC) and the Welch Foundation (TWU))