Theoretical Study On Microscopic Mechanism And Regulation Of Luminescence For Fluorescent Protein Mutants

Biomacromolecules are the basic substances that constitute life,including proteins,phospholipids and nucleic acids.Most of the life activities and functions occurring in living organisms are completed with the participation of biological macromolecules,and previous studies have clearly shown that these life activities and functions are closely related to the structure of the macromolecular system and its microscopic dynamics.In recent decades,structural biologists have developed a large number of novel analytical techniques to pursuit high-resolution of three-dimensional structures in different conformational states for biological macromolecules.More and more three-dimensional structure of biological macromolecular systems with near-atomic and even atomic resolution,and these achievements which greatly promotes people's understanding of the life activities and corresponding functions for the micro-scale biological system.Among these microscopic resolution techniques,the genetically encoded unnatural amino acid technology is indeed a breakthrough in progress.The maturity of this technology has led to the development of more and more protein structures with various physical,chemical and biological properties after being transformed by non-natural amino acids.Although the micro-analysis technology has made great progress,it is still limited by the bias of the experimental means in terms of temporal and spatial resolution,and it is often impossible to effectively capture and describe the micro-dynamic process of the biomacromolecule system.However,since the 1990 s,the rapid development of computational abilities and simulation technologies provides a solid and reliable microscopic physical image for the microscopic dynamics of biological macromolecular systems and the theoretical study of corresponding biological functions,provide a new approach to describe the microscopic dynamic process and then play a perfect supplementary for experimental methods in biochemistry research.At present,the combination of computer simulation methods and experimental methods has became a commonly used method for developing microscopic dynamics of biological macromolecular systems.At the same time,various computer simulation methods have been rapidly developed for different research demands,including molecular dynamics simulation,quantum chemical calculation,molecular modeling,molecular electronic spectroscopy and other computer simulation techniques.Through comparing with the experimental characterization results,the theoretical simulation can not only analyze the physical images implied in the experimental data,but also provide content that cannot be resolved by the experiment.The development of theoretical simulation,therefore,can effectively assist the experiment to deep analysis the characterization results and provide new microscopic physical pictures.Conversely,experimental characterization data can also significantly contribute to improve the theoretical model's deficiencies,thus promote theortical simulations provide more accurate physical pictures for micro-dynamic process research.Based on well developed simulation theory and tools,in this paper,I focus on studying several microscopic kinetic mechanisms for proteins in which perform important biological functions within organisms,such as manganese superoxide dismutase and fluorescent protein.The manganese superoxide dismutase is an important active oxygen scavenging enzyme in mitochondria.And the fluorescent protein plays a key role in the field of biological detection.Through theoretical simulation and experimental methods,we successfully analyze the microscopic mechanism of acetylation mutation modulation of manganese superoxide dismutase activity at specific sites.Combining theoretical studies on the relationship between structural features of fluorescent proteins and corresponding emission spectra at the molecular level,I further successfully design a series of chromophores with potentially make significant red shift in the emission spectrum,which greatly expand the scope of experimental research.Moreover,the spatial range of the fluorescent protein spectrum provides not only excellent candidates but also a set of procedures and guidelines for predicting and evaluating novel fluorescent protein chromophores.Furthermore,through the preliminary theoretical study of the long-range electron transfer process that induce fluorescence quenching in the green fluorescent protein mutant system,theoretically reveals that the electron transfer pathway of the system with high probability and the micro-modulation mechanism of the electron transfer coupling constant change in the molecular dynamic simulation.The structure of this paper is as follows:In the first chapter,the background and introduction of this paper are introduced,including the introduction of superoxide dismutase and fluorescent protein.In the second chapter,we briefly introduces the main theoretical background knowledge of the micro-dynamics mechanism of protein system,especially the microscopic mechanism and regulation of luminescence for fluorescent protein mutants,including molecular dynamics simulation,quantum chemistry,protein electrostatic interaction and molecular electron spectroscopy.In the third chapter,cooperates with the experiment,I successfully carry out theoretical study on microscopic kinetic process of the wild-type manganese superoxide dismutase in the mitochondria and lysine acetylation mutation at position 68 by applying the combination of molecular dynamics simulation and the Poisson-Boltzmann equation.Based on theoretical analysis of the micro-kinetics for both manganese superoxide dismutase,I successfully reveal the intrinsic reasons for acetylation to effectively regulate manganese superoxide dismutase activity.Molecular dynamics simulation clearly shows that the lysine acetylation at position 68 does only slightly modify the structure of manganese superoxide dismutase.The manganese superoxide dismutase before and after the acetylation mutation,thus are ahieved by solving the Poisson-Boltzmann equation.The electrostatic interaction of the solvent-contactable surface of superoxide dismutase clearly reveals that the lysine acetylation mutation at position 68 induces the positive charge content of the protein interface around the manganese active site of manganese superoxide dismutase are significantly reduced.The diffusing superoxide anion into the active space reaction,thence,is difficult to reach as compared to those before the acetylation mutation.The internal mechanism of the change of manganese superoxide dismutase activity caused by the acetylation mutation at position 68,is therefore successfully explained.In the fourth chapter,combining the molecular dynamics simulation with time-dependent density functional theory and QM/MM theory,I propose the theoretical research on the red shift phenomenon of different fluorescent protein emission spectra at the molecular level,and successfully design a series of chromophore group which could induce a significant red shift in the emission spectrum.On the theoretical level,the red shift phenomenon of the emission spectrum in the fluorescent protein is correlated and corresponding to the structural features of the chromophore group.Therefore,the microscopic mechanism of luminescence for fluorescent protein is also described,and the luminescence of fluorescent protein is regulated.Based on the current six fluorescent protein chromophores with well-defined experimental results,I carefully benchmark present commonly used calculation methods,and find that the TD-?-B97XD/6-311+G(2d,2p)quantitative calculation method combines with the implicit solvation model and a dielectric constant of 4 is the most suitable model for the emission spectrum in our studied system.Therefore,in the subsequent calculations,this simulation method is applied to study the effects of different structural components in fluorescent protein chromophore on its corresponding emission spectrum,including the degree of conjugation,the type of substituents,the position of the substituents,and the number of substituents.Based on the combination of these theoretical studies,I successfully designed several chromophore mutants that are expected to induce red shift of the emission spectrum more than 60 nm.In addition,by applying molecular dynamics simulation,I also evaluate the structural stability of the designed chromophore group in the fluorescent protein system.Moreover,by applying QM/MM theory,I also carefully study the emission spectra of the newly designed fluorescent protein complex fromed by red fluorescent protein with the newly designed chromophore.In the fifth chapter,combining with molecular dynamics simulation,density functional theory,Marcus electron transfer theory and Pathways theoretical model,I carry out a preliminary theory on the single-step direct and multi-step electron transfer process that indude fluorescence quenching in different green fluorescent protein mutants.The study clearly reveals that the electron transfer pathway with high probability,the maximum electron transfer effective coupling constant and the miscroscopic mechanism for regulating the change of coupling constant in molecular dynamic simulation.Based on the donor and acceptor residue molecules during electron transfer achieved above,the atoms that make the dominant contribution to the donor and acceptor residues in electron transfer process have been extracted by natural orbital analysis and specific orbital atom contribution analysis.Furthermore,by applying the Pathways model,I successfully carry out the study of searching the electron transfer pathway of the system in the molecular dynamics trajectory and the system's electron transfer pathway with high probability.The effective coupling constant distribution are also achieved in this way.Finally,by applying the cluster and principal component analysis of molecular dynamics simulation trajectories,I find that the mechanisms responsible for the modulation coupling constant changes in different mutants are exactly different.Moreover,the change in coupling constants of some varients is caused by the change in the distance beween the electron donor and acceptor due to its low-frequency atomic displacement.In the sixth chapter,a brief summary of all the research work,and also a brief introduction to the ongoing research work,including the study of the dynamic mechanism of membrane proteins in the process of biofilm conformational changes and electron mechanisms in specific green fluorescence protein mutants.