Studying Temperature Dependence Of Protein Dynamics By Using Quasi-elastic Neutron Scattering

Proteins are the engine of life and conduct most bio-functions in life.There are millions of proteins on the planet with different structures,and they perform various biological functions.Understanding and predicting proteins' function solely based on their structures are often misleading.For example,proteins with similar structures may have distinct functions,while proteins with different structures may perform similar functions.This is because protein dynamics plays an indispensable role in determining protein function.Studying protein dynamics will furthers the understanding of various life activities,which will promote the development of medicine and healthcare.Additionally,it will also unravels the microscopic mechanisms governing the protein stability and catalytic activity,crucial for the biotechnology.However,it's difficult to study protein dynamics due to the marginal stability and complexity.Neutron scattering covers a broad temporal and spatial windows,which span from sub picoseconds to hundreds of nanoseconds,and from sub angstrom to tens of nanometers.Moreover,as neutron is highly sensitive to hydrogens,one can study any desired region or component by deuterating the rest of the system.These unique features render the neutron scattering of great power in revealing dynamics in proteins.Here we study temperature dependence of protein dynamics by using quasi-elastic neutron scattering,including the protein dynamical transition at ~200 K and protein unfolding from 300 K to 363 K:1.Proteins with different structures show the dynamical transition at a similar temperature ~200 K: below this temperature,the protein mainly exhibits a simple harmonic vibration,above which the protein softens and exhibits anharmonic motion.Below this temperature,some proteins not only lose their freedom of anharmonic motion,but also lose biological activity.In the past three decades,many experimental and theoretical works have ascribed this dynamical transition to the water molecules on protein surface,which rationalize why the dynamical transition temperatures of different proteins are similar.However,by combining quasi-elastic neutron scattering and perdeuterated protein,we found that even a lyophilized protein can show a dynamical transition at ~200 K.This result indicates that the dynamical transition is independent of water and is an intrinsic property of proteins,but the introduction of water might greatly enhance this transition.This discovery has raised challenges on the classical picture over the past three decades that the dynamical transition in the protein is caused by its surface water,and may lead to further research on this topic.Furthermore,combining quasi-elastic neutron scattering instruments with different time resolutions and dielectric spectroscopy,we realized that the dynamical transition of dry perdeuterated proteins at ~200 K results from unfreezing of the relaxation of the protein structures on the laboratory equilibrium time(100-1000 s),which softens the entire bio-macromolecules.2.Protein macromolecules are generally not resistant to high temperatures(denature at ~50 °C),which limits their use in enzyme engineering and rigorous mechanism studies.Therefore,understanding the stratergy of protein stabilization at high temperature,and improve the thermostability of proteins has drawn great attentions of both scientific community and industry.Enzyme engineering are often designed to increase the intramolecular forces inside the protein molecules,e.g.,introducing hydrogen bonds,salt bridges and hydrophobic interactions between protein residues,which enhance the thermostability in terms of the enthalpic effect.We compared the temperature dependent dynamics of a mesophilic protein CYP101A1 and a thermophilic protein CYP119 in the cytochrome P450 family by using quasi-elastic neutron scattering and nuclear magnetic resonance spectroscopy.An unexpected phenomenon that the thermophilic CYP119 showed higher structural flexibility than the mesophilic CYP101A1 rendered us a great surprise.Thus,we used the fluorescence technique to measure the thermodynamic curves of the two proteins,i.e.,the Gibbs free energy as a function of temperature.The analyses on their thermodynamic curves showed that CYP119 is actually more unstable in terms of enthalpy due to its smaller inter-residue interaction energy.The origin of its higher thermostability arises from the less entropy change of the protein during the denaturing process,i.e.,less driving force towards denaturation.The less entropy change may be attributed to the greater structural flexibility of the protein at the native state.Contrary to the conventional "enthalpy enhancement" mechanism,this study found that the "high entropy" at the native state can reduce the driving force of “entropy gain” in the denaturing process,thus raising the protein's thermostability.Meanwhile the "high entropy" state might also enhance the enzymatic activity of the bio-macromolecules.The results provide a new idea for the design of thermostable proteins,which may raise both proteins' thermostability and enzymatic activity by raising the flexibility of the proteins at the native folded state.In summary,this manuscript systematically studied the protein dynamical transition at low temperature(~200K)and protein unfolding at high temperature(300K to 363K)by taking advantage of quasi-elastic neutron scattering.The investigations on these two physical processes not only explores the physical mechanism of protein dynamics at different temperatures,but also demonstrates the great potential of quasi-elastic neutron scattering in protein dynamics research.