PROJECT SUMMARY
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.
