Energetics of Intermolecular Hydrogen Bonds in a Hydrophobic Protein Cavity

Lan Liu, Alyson Baergen, Klaus Michelsen, Elena N. Kitova, Paul D. Schnier, John S. Klassen

This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the −7 charge state) of complexes of bovine β-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)(7–) ions, compared with that of the (Lg + PA)(7–) ion, it is concluded that the –OH groups of the X-OHPA ligands participate in strong (5 – 11 kcal mol(–1)) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the –OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the –OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (Ka) measured for 7-OHPA [(2.3 ± 0.2) × 10(5) M(–1)] is similar to the value for the PA [(3.8 ± 0.1) × 10(5) M(–1)]; Ka for 3-OHPA [(1.1 ± 0.3) × 10(6) M(–1)] is approximately three-times larger, whereas Ka for 16-OHPA [(2.3 ± 0.2) × 10(4) M(–1)] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand –OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.