What it is
A multiplexed method uses intact-protein hydrogen-deuterium exchange mass spectrometry to measure the energies of rare, higher-energy conformational fluctuations across 5,778 protein domains of 28-64 amino acids in length, in parallel. The data reveal hidden variation in these fluctuations even between sequences that share the same fold and the same global folding stability. Site-resolved hydrogen-exchange NMR on 13 domains showed the fluctuations often unfold entire secondary-structure elements that are less stable than the fold as a whole, and computational modeling linked structural features to the measured fluctuations and guided mutations that stabilized the weak segments.
Why it matters
Native protein structures are now predictable with high accuracy, but the rare higher-energy states that govern function, interactions, aggregation, and immunogenicity have stayed largely invisible and unpredictable for lack of measurements at scale. This work converts those hidden fluctuation energies into a large quantitative dataset, supplying the kind of training data that machine-learning and physics-based models of protein energy landscapes have lacked.
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Filed underprotein folding, mass spectrometry, hydrogen-deuterium exchange, protein design, biophysics