Most biochemists and biophysicists would agree that ionic interactions are extremely important for molecular association of protein and nucleic acids. But it is not well known that the ionic interactions are highly dynamic. Through entropic contribution to the binding free energy, the dynamics of the ionic interactions play an important role in DNA recognition by proteins. Funded by the National Science Foundation (NSF) and the National Institutes of Health (NIH), our group has spent a decade to study how ions and ionic moieties behave at an atomic level in protein-DNA association processes.
Using nuclear magnetic resonance (NMR) spectroscopy as our primary tool for research, we study how counterions behave in the protein-DNA association. The large net charge on the surface of nucleic acids electrostatically attract and condense cations, creating a zone called the ion atmosphere. Experimental approaches for quantitative investigations of cations in the ion atmosphere have been developed. The counterions were found to rapidly diffuse within the ion atmosphere. Some of the counterions are released from the ion atmosphere when nucleic acids bind to proteins, neutralizing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had only indirectly been implicated by salt-concentration dependence of the equilibrium constants for molecular association. Recently, the direct detection of the release of counterions has become possible through spectroscopic observation of ions. This allows more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein-nucleic acid association.
Using NMR and other biophysical methods, we also study the dynamic properties of ion pairs of protein side chains and DNA phosphates. The ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact. This state is called a contact ion pair (CIP). In the other major state, the cation and the anion are intervened by water. This state is called a solvent-separated ion pair (SIP). Transitions between CIP and SIP states rapidly occur at the molecular interfaces. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Compared to Lys side chains, Arg side chains exhibit a higher propensity to directly interact with nucleotide bases, partly due to stronger cation-p interactions and a smaller desolvation energy. Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play thermodynamic and kinetic roles in protein-nucleic acid interactions.
Selected publications from this research project
Pletka, C.C., Nepravishta, R., Iwahara, J. (2020) Detecting counterion dynamics in DNA-protein association. Angew Chem Int Ed 59, 1465-8.
Yu, B., Pettitt, B.M., Iwahara, J. (2019) Experimental evidence of solvent-separated ion pairs as metastable states in electrostatic interactions of biological macromolecules. J Phys Chem Lett 10, 7937-41.
Esadze, A., Chen, C., Zandarashvili, L., Roy, S., Pettitt, B.M., Iwahara, J. (2016) Changes in conformational dynamics of basic side chains upon protein-DNA association. Nucleic Acids Res 44, 6961-70.
Chen, C., Esadze, A., Zandarashvili, L., Nguyen, D., Pettitt, B.M., Iwahara, J. (2015) Dynamic equilibria of short-range electrostatic interactions at molecular interfaces of protein-DNA complexes. J Phys Chem Lett 6, 2733-7.
Anderson, K.M., Esadze, A., Manoharan, M., Brüschweiler, R., Gorenstein, D.G., Iwahara, J. (2013) Direct observation of the ion-pair dynamics at a protein-DNA interface by NMR spectroscopy. J Am Chem Soc 135, 3613-9.