Biomolecular Electrostatics by NMR Spectroscopy

In biological chemistry, electrostatics plays a crucial role in molecular recognition and catalysis by proteins and nucleic acids. Accurate information about electrostatic interactions is essential for the success of drug development and protein engineering. However, studying these interactions, particularly those involving mobile charges, presents significant challenges. In biological systems, mobile charges are carried by ions, metabolites, or highly flexible regions of biological macromolecules. The overall goal of our renewed project is to enhance the applicability of our nuclear magnetic resonance (NMR) methods for investigating biomolecular electrostatics. We will focus on the following two specific objectives: 1) to refine NMR methods for direct measurements of electrostatic potentials and 2) to expand NMR methods for investigating ions in the vicinity of biomolecules. For Objective 1, we will develop new paramagnetic NMR techniques to measure the local electrostatic potentials in intrinsically disordered regions (IDRs) of proteins. We will use the experimentally measured electrostatic potentials to derive realistic structural ensembles of proteins that include both IDRs and folded domains. The NMR-based methods to directly measure electrostatic potentials will also be applied to noncanonical DNA such as G-quadruplex and i-motif structures. To address Objective 2, we will utilize custom-made diffusion NMR probe hardware designed to produce strong field gradients, enabling diffusion measurements of ions such as potassium, magnesium, calcium, chloride, and sulfate ions in biomolecular solutions. By analyzing the diffusion NMR data, we will quantify the counterions released from each macromolecule upon forming a complex and estimate the increase in entropy resulting from this counterion release. Through the development of novel methods using NMR spectroscopy, our project will help advance the field of biomolecular electrostatics.