Nmr Investigation of Energy Barriers for Hydrogen-Bond Breakage of Protein Side-Chain NH3+ Groups

Abstract

Hydrogen bonds and ion pairs involving charged side chains are important for protein function. However, their dynamic properties are not well understood. Recently we demonstrated that lysine side-chain NH3+ groups are extremely useful probes for NMR investigations of the hydrogen-bond/ion-pair dynamics. Previously we found that bond rotations of protein side-chain NH3+ groups are nearly as rapid as those of side-chain CH3 groups despite the presence of hydrogen bonds. To understand this phenomenon, we have studied energy barriers for NH3+ rotations requiring the transient breakage of hydrogen bonds. For the HoxD9 homeodomain-DNA complex, we investigated the temperature dependence of the internal motions of lysine side-chain NH3+ groups forming intermolecular ion pairs with DNA. For these NH3+ groups, we determined order parameters and correlation times for bond rotations and reorientations at four temperatures. The order parameters were virtually independent of temperature in this range. In contrast, the NH3+ bond-rotation correlation times were found to depend strongly on temperature. Based on transition state theory, the energy barriers for NH3+ rotations were analyzed and compared to those for CH3 rotations. Enthalpies of activation for NH3+ rotations were found to be significantly higher than those for CH3 rotations, which can be attributed to the requirement of hydrogen-bond breakage. However, the transition states in which hydrogen bonds with water molecules should be transiently broken are entropically favorable, and the overall free energies of activation for NH3+ rotations are as low as those for CH3 rotations. This entropic reduction in energy barriers can accelerate molecular processes requiring hydrogen bond breakage and may be kinetically important for protein function. This work was supported by Grant CHE-1307344 from the National Science Foundation.