Exploring structure and stability in ion binding domains with quantum cascade laser infrared spectroscopy

We use quantum cascade laser (QCL)-based infrared spectroscopy to probe the structure and dynamics of ion binding sites at the heart of biological signaling processes. Our results suggest how ion occupancy, ion substitution, and mutations within or in the vicinity of ion binding domains modulate not only the equilibrium configurations of their host structures, but also the potential energy landscapes explored by these molecules as they bind and release signaling species such as Ca2+. As such, this class of experiments maps the evolution of ion signaling structures as they carry out their normal biological functions, or - as in the case of disease-associated mutants - give rise to pathological consequences.

Selective and dynamic ion binding regulates nearly all aspects of cellular metabolism. For example, signaling proteins respond to millisecond transients in [Ca2+] while rejecting the effects of other ions in millionfold excess. Mutations that perturb the ion binding domains of these biomolecules are disproportionately associated with a range of diseases. Nevertheless, characterization of binding site architectures often remains limited to static crystallographic structures, and – even for the most-studied signaling proteins – large gaps remain in knowledge of the conformational sequences leading from ion binding to downstream signal transduction. Our research harnesses both equilibrium and time-resolved infrared spectroscopy to elucidate these structural and dynamic factors in ion binding structures including EF-hand Ca2+ sensors and lanthanide binding tags.