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

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

Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egr‑1 Zinc-Finger Protein with DNA

The transcription factor Egr-1 specifically binds as a monomer to its 9 bp target DNA sequence, GCG­T­G­G­GCG, via three zinc fingers and plays important roles in the brain and cardiovascular systems. Using fluorescence-based competitive binding assays, we systematically analyzed the impacts of all possible single-nucleotide substitutions in the target DNA sequence and determined the change in binding free energy for each. Then, we measured the changes in binding free energy for sequences with multiple substitutions and compared them with the sum of the changes in binding free energy for each constituent single substitution. For the DNA variants with two or three nucleotide substitutions in the target sequence, we found excellent agreement between the measured and predicted changes in binding free energy. Interestingly, however, we found that this thermodynamic additivity broke down with a larger number of substitutions. For DNA sequences with four or more substitutions, the measured changes in binding free energy were significantly larger than predicted. On the basis of these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of such sequences that might functionally sequester Egr-1

Dynamic Equilibria of Short-Range Electrostatic Interactions at Molecular Interfaces of Protein–DNA Complexes

Intermolecular ion pairs (salt bridges) are crucial for protein–DNA association. For two protein–DNA complexes, we demonstrate that the ion pairs of protein side-chain NH₃⁺ and DNA phosphate groups undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. While the crystal structures of the complexes show only the solvent-separated ion pair (SIP) state for some interfacial lysine side chains, our NMR hydrogen-bond scalar coupling data clearly indicate the presence of the contact ion pair (CIP) state for the same residues. The 0.6-μs molecular dynamics (MD) simulations confirm dynamic transitions between the CIP and SIP states. This behavior is consistent with our NMR order parameters and scalar coupling data for the lysine side chains. Using the MD trajectories, we also analyze the free energies of the CIP–SIP equilibria. This work illustrates the dynamic nature of short-range electrostatic interactions in DNA recognition by proteins

Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling

The residence times of molecular complexes in solution are important for understanding biomolecular functions and drug actions. We show that NMR data of intermolecular hydrogen-bond scalar couplings can yield information on the residence times of molecular complexes in solution. The molecular exchange of binding partners via the breakage and reformation of a complex causes self-decoupling of intermolecular hydrogen-bond scalar couplings, and this self-decoupling effect depends on the residence time of the complex. For protein–DNA complexes, we investigated the salt concentration dependence of intermolecular hydrogen-bond scalar couplings between the protein side-chain ¹⁵N and DNA phosphate ³¹P nuclei, from which the residence times were analyzed. The results were consistent with those obtained by ¹⁵N_<i>z</i>-exchange spectroscopy. This self-decoupling-based kinetic analysis is unique in that it does not require any different signatures for the states involved in the exchange, whereas such conditions are crucial for kinetic analyses by typical NMR and other methods