Detecting protein fluctuations at scale

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Protein structures and structure prediction methods generally capture a static, low-energy state of a protein. However, proteins dynamically fluctuate between different conformations, including high-energy conformations that can influence protein function and interactions. Experimentally characterizing high-energy states is challenging, limiting efforts to uncover general principles of protein energy landscapes.

Now, Ferrari et al. have developed a multiplexed hydrogen–deuterium exchange mass spectrometry approach for large-scale profiling of protein energy landscapes. The approach is based on mixing hundreds of small domains into D2O and analyzing the deuterium incorporation into each domain at many timepoints using liquid chromatography–ion mobility mass spectrometry. An automated computational pipeline converts these hydrogen-exchange measurements into distributions of residue opening energies for each protein. These opening energy distributions reveal fluctuations to high-energy ‘partially open’ states in which subsets of residues become exposed to solvent, as well as the energy of unfolding the entire protein (global folding stability, ΔGunfold). Importantly, domains with similar native structures and similar global folding stabilities often had very different energies of fluctuations, highlighting the value of quantifying the fluctuations for each individual domain.