Use of High Pressure NMR Spectroscopy to Rapidly Identify Proteins with Internal Ligand-Binding Voids

Small molecule binding within internal cavities provides a way to control protein function and structure, as exhibited in numerous natural and artificial settings. Unfortunately, most ways to identify suitable cavities require high-resolution structures a priori and may miss potential sites. Here we address this limitation via high-pressure solution NMR spectroscopy, taking advantage of the distinctive nonlinear pressure-induced chemical shift changes observed in proteins containing internal cavities and voids. We developed a method to rapidly characterize such nonlinearity among backbone ¹H and ¹⁵N amide signals without needing to have sequence-specific chemical shift assignments, taking advantage of routinely available ¹⁵N-labeled samples, instrumentation, and 2D ¹H/¹⁵N HSQC experiments. From such data, we find a strong correlation in the site-to-site variability in such nonlinearity with the total void volume within proteins, providing insights useful for prioritizing domains for ligand binding and indicating mode-of-action among such protein/ligand systems. We suggest that this experimental approach is a rapid and useful probe of otherwise hidden dynamic architectures of proteins, providing novel insights and opportunities into ligand binding and control.