Thermodynamic evaluation of ligands binding to the Grb2 SH2 domain: effects of α,α-disubstitution at the pY+1 position
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A series of phosphotripeptide ligands for the Grb2 SH2 domain was designed and synthesized, each of which derived from the minimal consensus sequence required for binding: Ac-pYXN. The binding affinity and related thermodynamic parameters were determined by isothermal titration calorimetry. Both the size and connectivity of the side-chain was varied. The consequences of incorporating α,α-disubstitution at the pY+1 residue on binding thermodynamics were evaluated, as were the effects of constraining the side-chains in a ring. The series was evaluated from a number of perspectives: (1) increasing size of the pY+1 residue by utilizing various amino acid types: monoalkyl, dialkyl, or cycloalkyl; (2) comparisons between ligands with the same number of carbons (scission control); and (3) by comparing ligands incorporating cyclic pY+1 residues with those incorporating α,α-dialkyl residues with one fewer methylene group (excision control). Inconsistencies in the thermodynamic consequence of constraining the backbone were observed within this set of ligands, which reveal the limitations of our understanding of protein-ligand interactions. Aspects of both the classical and non-classical hydrophobic effect were observed, but the occurance of one over the other could not be explained.