Physicochemical Properties of Ion Pairs of Biological Macromolecules

Ion pairs (also known as salt bridges) of electrostatically interacting cationic and anionic moieties are important for proteins and nucleic acids to perform their function. Although numerous three-dimensional structures show ion pairs at functionally important sites of biological macromolecules and their complexes, the physicochemical properties of the ion pairs are not well understood. Crystal structures typically show a single state for each ion pair. However, recent studies have revealed the dynamic nature of the ion pairs of the biological macromolecules. Biomolecular ion pairs undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. This dynamic behavior is reasonable in light of the fundamental concepts that were established for small ions over the last century. In this review, we introduce the physicochemical concepts relevant to the ion pairs and provide an overview of the recent advancement in biophysical research on the ion pairs of biological macromolecules

Discrete-State Kinetics Model for NMR-Based Analysis of Protein Translocation on DNA at Equilibrium

In the target DNA search process, sequence-specific DNA-binding proteins first nonspecifically bind to DNA and stochastically move from one site to another before reaching their targets. To rigorously assess how the translocation process influences NMR signals from proteins interacting with nonspecific DNA, we incorporated a discrete-state kinetic model for protein translocation on DNA into the McConnell equation. Using this equation, we simulated line shapes of NMR signals from proteins undergoing translocations on DNA through sliding, dissociation/reassociation, and intersegment transfer. Through this analysis, we validated an existing NMR approach for kinetic investigations of protein translocation on DNA, which utilizes NMR line shapes of two nonspecific DNA–protein complexes and their mixture. We found that, despite its use of simplistic two-state approximation neglecting the presence of many microscopic states, the previously proposed NMR approach provides accurate kinetic information on the intermolecular translocations of proteins between two DNA molecules. Interestingly, our results suggest that the same NMR approach can also provide qualitative information about the one-dimensional diffusion coefficient for proteins sliding on DNA

The structure of the Dead ringer–DNA complex reveals how AT-rich interaction domains (ARIDs) recognize DNA

The AT-rich interaction domain (ARID) is a DNA-binding module found in many eukaryotic transcription factors. Using NMR spectroscopy, we have determined the first ever three-dimensional structure of an ARID–DNA complex (mol. wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster. ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non-canonical helix–turn–helix motif, and through a concomitant structural rearrangement that produces stabilizing contacts from a β-hairpin. Dead ringer’s preference for AT-rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine–thymine base step. Amino acids that dictate binding specificity are not highly conserved, suggesting that ARIDs will bind to a range of nucleotide sequences. Extended ARIDs, found in several sequence-specific transcription factors, are distinguished by the presence of a C-terminal helix that may increase their intrinsic affinity for DNA. The prevalence of serine amino acids at all specificity determining positions suggests that ARIDs within SWI/SNF-related complexes will interact with DNA non-sequence specifically

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