Examining g-quadruplex metal site heterogeneity and the influence of peptide binding using 2D IR spectroscopy

The National Institute of General Medical Sciences (NIGMS) awarded a $371,250 Project Grant under the Biomedical Research and Research Training (CFDA 93.859) program to Southern Illinois University Carbondale (SIU) to conduct research on the structural and dynamic properties of G-quadruplex (G4) nucleic acid motifs and their interactions with regulatory proteins.

The project aims to use advanced two-dimensional infrared (2D IR) spectroscopy techniques, along with site-specific labeling, to probe the heterogeneity of metal ion binding sites within G4 structures and how this impacts their folding and unfolding behaviors and ability to interact with stabilizing or unwinding peptides derived from G4-binding proteins. The findings from this research are expected to provide fundamental insights into the physicochemical properties of G4s and inform the development of drugs and imaging probes that target G4-protein complexes. The project also supports the training of graduate and undergraduate students in this interdisciplinary research area.

Abstract

G-quadruplexes (G4S) are four-stranded nucleic acid motifs that have been implicated in a diverse array of biological functions and diseases and have emerged as attractive targets for drug design. The coordination of alkali metal ions (e.g., k+, NA+) to guanine C6=o carbonyls is a critical factor in their assembly and stabilization, and thus, is likely to influence interactions with proteins that recognize and remodel G4S to perform regulatory functions. Although thousands of G4S have been structurally characterized and there is extensive understanding of their in vitro folding/unfolding behaviors, the overall lack of molecular-level data about their interactions with proteins results in a major GAP in understanding if/how metal binding and protein interactions are coupled. Ultrafast two-dimensional infrared (2D IR) spectroscopy is a powerful probe of local electrostatics, vibrational coupling, and femtosecond-picosecond fluctuations. Together with site specific labeling, IT has been applied to many proteins to discover subtle variations in binding interactions that are often hidden from high-resolution techniques. We have recently developed 2D IR methods for structural analysis of G4S and showed that isotope editing can detect variations in metal site occupancy, plasticity, and dynamics. Combined, 2D IR provides a useful method to probe the interplay between metal coordination, folding/unfolding, and protein binding in G4S. This proposal expands on established methods in two specific aims. In the first aim, we use 2D IR and isotope editing to test the hypothesis that native state dynamics and C6=o bond frequencies correlate with the global stability of a G4, sequential assembly and disassembly mechanisms, and native state metal exchange rates. In the second aim, we examine the interactions of G4S with peptides derived from proteins that either stabilize or unwind G4S and test the hypothesis that peptide (protein) binding biases the metal stabilized G4 core in the direction of unfolding or folding depending on function. If this study is successful, IT will provide fundamental insight into the physicochemical properties of G4S and may provide clues for how to target G4:protein complexes with drugs and imaging probes. The project also provides an interdisciplinary training environment for graduate and undergraduate students that will prepare them for future careers in research.