Exploring the physics of proteins at molecular level by neutron and X-ray scattering

Protein conformational dynamics are believed to ultimately govern the biological activities and functions of proteins. Hence, a deeper understanding of the protein dynamics is crucial for elucidating the structural pathways or the transition mechanisms necessary for regulating the physical and chemical processes. The direct correlation of a wide range of protein dynamics to function still remains unclear, posing a major challenge to biophysical community. In this dissertation, the relationships among the protein's conformation, dynamics and function are investigated using the state-of-the-art neutron and X-ray scattering techniques. Taking the advantage of comparable wavelength or momentum of neutron and X-ray to that of the atoms within biomolecules, we studied the protein dynamics at the molecular level over the timescale of a few femtoseconds to nanoseconds regime. Our results demonstrate that the protein dynamic behavior is similar to that of glass forming liquids, where the relaxation process is non-exponential and the collective excitations are highly damped. Speci cally, picosecond to nanosecond dynamics, also known as beta-relaxation process decays logarithmically over the time. Remarkably, such dynamic phenomena revealed the direct experimental evidences of structure-dynamics-function relationship of a large variety of protein family, such as a large hyperthermophilic protein, a membrane protein, and the native and denatured globular proteins. First, we used quasi-elastic neutron scattering (QENS) to study the dynamics of a hyperthermophilic protein from the deep-sea on the timescale of picosecond to nanosecond, and revealed that the dynamic property of a mesophilic protein is largely affected by the high pressure and temperature. Speci cally, high pressure distorts the protein energy landscape and therefore the activity, while the hyperthermophilic protein restraints such effects. Next, the mechanisms of light activation of a G-protein-coupled receptor (GPCR) prototype, rhodopsin, were studied using small-angle neutron scattering (SANS) and QENS. The SANS data indicated the large conformational change in rhodopsin upon photoactivation; the QENS results revealed the signi cant difference in the intrinsic protein dynamics between the dark-state rhodopsin and the ligand-free apoprotein, opsin. These observed conformational and dynamical differences in rhodopsin upon photoactivation are due to the influence of the covalently bound retinal chromophore. Eventually, we successfully applied the concept of generic energy landscape based upon the dynamic behavior possessed by the proteins to explain their activities. In the third project, the phonon-like collective excitations in proteins were investigated using inelastic neutron and X-ray scattering techniques. Such excitations correspond to the intrinsic protein dynamics necessary to overcome the conformational barriers, crucial for enzyme catalysis and ligand-binding. Our data show the apparent softening of protein with a rise in temperature, corresponding to the protein conformational flexibility. Speci cally, our results suggest that the native globular protein balances the protein conformational flexibility and rigidity for the biological activity. Lastly, we used small-angle X-ray scattering (SAXS) to study the conformational change in periplasmic ligand-binding protein (PBP) upon bound to peptide. The three-dimensional shape reconstruction of a periplasmic protein MppA computed from SAXS intensity profi le using ab-initio modeling perfectly matches its crystal structure.