Temperature Dependence of Internal Motions of Protein Side-Chain NH₃⁺ Groups: Insight into Energy Barriers for Transient Breakage of Hydrogen Bonds

Abstract

Although charged side chains play important roles in protein function, their dynamic properties are not well understood. Nuclear magnetic resonance methods for investigating the dynamics of lysine side-chain NH₃⁺ groups were established recently. Using this methodology, we have studied the temperature dependence of the internal motions of the lysine side-chain NH₃⁺ groups that form ion pairs with DNA phosphate groups in the HoxD9 homeodomain–DNA complex. For these NH₃⁺ groups, we determined order parameters and correlation times for bond rotations and reorientations at 15, 22, 28, and 35 °C. The order parameters were found to be virtually constant in this temperature range. In contrast, the bond-rotation correlation times of the NH₃⁺ groups were found to depend strongly on temperature. On the basis of 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, entropies of activation substantially reduce the overall free energies of activation for NH₃⁺ rotations to a level comparable to those for CH₃ rotations. This entropic reduction in energy barriers may accelerate molecular processes requiring hydrogen bond breakage and play a kinetically important role in protein function