Following the Complete Thermal Landscape of a Protein by Neutron Scattering

Protein dynamics is intimately coupled to hydration and stability, yet direct observation of the full sequence of thermally induced transitions remains instrumentally challenging. Here, we present a study that exploits the unique properties of Yfh1, one of the few natural proteins that undergoes both cold and heat denaturation at experimentally accessible temperatures, to probe protein dynamics by advanced elastic incoherent neutron-scattering instrumentation that allows detection of motions over an unprecedently broad momentum-transfer range and a range of 100-330 K temperatures. We identified three distinct dynamical regimes, corresponding to the protein dynamical transition, cold denaturation, and heat denaturation. We could distinguish motions associated predominantly with the protein from those of its hydration shell. Importantly, our work also provides what is, to our knowledge, the first direct neutron-scattering observation of the dynamical consequences of cold denaturation in a protein. Hydration water displays a marked anomaly near 260 K that disappears following heat denaturation, revealing a strong coupling between native protein structure and water dynamics. These results establish a unified dynamical picture of protein stability across its complete thermal landscape.