Detecting protein fluctuations at scale

Proteins dynamically fluctuate between different conformations, including high-energy states that can influence protein function and interactions. Experimentally characterizing these high-energy states is challenging, limiting efforts to uncover general principles of protein energy landscapes. The authors have developed an approach to quantify protein energy fluctuations. This approach involves mixing small domains into D2O and analyzing deuterium incorporation into each domain at multiple timepoints using liquid chromatography–ion mobility mass spectrometry. An automated computational pipeline converts these measurements into distributions of residue opening energies for each protein, revealing fluctuations to high-energy 'partially open' states and the energy of unfolding the entire protein. The approach highlights the value of quantifying fluctuations for each individual domain, even when domains have similar native structures and global folding stabilities.