Direct experimental characterization of contributions from self-motion of hydrogen and from interatomic motion of heavy atoms to protein anharmonicity

One of challenges in biophysics is to understand the connection between protein dynamics and its function. The challenge partially arises from the fact that protein present a variety of local atomic motions and collective dynamics on the same time scales, and this has rendered difficult the experimental identification and quantification of different dynamic modes. Here, taking lyophilized protein as examples, we combined the deuteration technique and the neutron scattering experiment to separate the self-motion and the collective interatomic motion of heavy atoms in proteins. We found that the self-motions of protein hydrogen atoms present a resolution-time-dependent anharmonic onset, with the onset temperature increasing when decreasing the resolution time. This can be ascribed to the thermal activation of local side-group motions, mostly the methyl rotations. In contrast, the collective dynamics of protein heavy atoms exhibit a resolution-time-independent anharmonicity around 200 K. Further dielectric spectroscopy and Brillouin light scattering results suggest that the anharmonicity of the heavy atoms results from unfreezing of the relaxation of the protein structures on the laboratory equilibrium time (100-1000 s), which softens the entire bio-macromolecules.