| Protein is vital component in all life being, which tightly involved with various life activities, it is key undertaker of life activities, and is material basis of life being. So undertaking protein research for further revealing the secret of life activities which is at the frontier of research in life science.The three-dimensional structure which encode with amino acids sequence is the basis of various biological functions. Knowledge of biological function of protein, we firstly need to learn the three-dimensional structure information. With the de-velopment of X-ray and NMR technique, one have accumulated a large amount of three-dimensional structure information, and yet, how the protein fold to specific three-dimensional structure from one-dimensional sequence remain be unclear. Previous s-tudies on protein folding mostly focused on simple proteins which usually have one single domain and can fold spontaneously. For these proteins, the folding typically fol-lows a two-state mechanism. Several folding theoretical models have been proposed to explain the experimental data of protein folding, including the diffusion-collision model, nucleation-condensation model, and so on. Particularly, the energy landscape theory provided a general framework for understanding the kinetics and thermodynam-ics properties of protein folding. With the development of experimental techniques and computing capability, the scope of protein folding studies have been extended to more complicated protein systems. Especially the folding of proteins contain cofactor. For such proteins, the folding behaviors can be largely regulated by the binding of cofac-tor, and show many new features. Due the limitation of the resolution of experimental methods, it is difficult to elucidate the folding mechanism by cofactor modulate at the molecular level. So it is necessary to study the relative problem by molecular dynamics method.For the classical all-atom molecular dynamics simulation, the number of system can reach up to hundreds of thousands of atoms, the time of simulation usually under microsecond in time scale. However, the time scale of many biological processes may reach up to millisecond second even longer. To solve the problem, one have proposed some methods to improve simulation efficiency, which included using coarse-graining the molecular representation or replica exchange method to increase conformational sample efficiency. These methods are efficiency for our work which involved with proteins contain cofactor.The thesis focused on effects of cofactor on protein folding and conformational distribution by coarse-grained method and efficiently conformational sample all-atom simulation methods. The thesis included follow three works:Study one:When the myoglobin execute the function of oxygen storage, which need the participation of heme group. Then, little is known about the role of myo-globin during its folding process. The experiments results showed that the heme group can improve the cooperatively of folding and structure stability of myoglobin, but the molecular mechanism is unclear. To investigate the regulatory mechanisms during myoglobin folding with heme group. In this work, we investigated the folding mecha-nism by coarse-grained method. The results showed that the apo-myoglobin exist a in-termediate state during folding process, is a low cooperative three-state folding. As far as intermediate state be concerned, the hydrophobic core have folded completely, the heme bind pocket haven’t folded yet. In the presence of heme group, the folding pro-cess of myoglobin showed high cooperative two-state mechanism, which accord with experimental results. The detail statistical results shown that the heme group binding obviously improve heme binding pocket moiety stability and folding rate, and build the interaction and coupled dynamics between heme binding pocket and hydrophobic core, strength the cooperatively of folding. The results profound insight into the molecular mechanism of protein folding and stability by heme group modulating.Study two:The protein folding energy landscape theory show that the folding mechanism is decided by three-dimensional topology structure. In recent years, the experimental results suggest, after the residues in Zn-ion binding site of Zn-finger pro-tein are replaced with hydrophobic core(or reverse method), the protein still can fold to same three-dimensional structure, but folding mechanism is obviously different. For example, the prokaryotic Zn-finger protein Ros87 and homologous zinc-lacking eu-karyotic protein Ml4 were characterized with recent experimental methods, and found the binding residues of zinc ion replaced hydrophobic residues can lead to fold mecha-nism from two-state mechanism to partly downhill folding mechanism, lower the coop-erativity of folding. In this work, we investigated the folding mechanism of Ros87 and M14 based on coarse-grained molecular dynamics simulation. The simulation results showed that the zinc-ion lacking M14 follow classical two-state folding mechanism; zinc-ion contain Ros87 follow partly downhill folding mechanism, which accord with experiment. Further analysis showed that, zinc-ion binding can improve nucleation point form, the Ros87 can further fold based on nucleation point, which accelerate the folding process; by contrast, even the protein M14 contain a hydrophobic core, the hydrophobic core form rely on stochastic collision, leading to a high potential barri-er exist between folding state and unfolding state. These results suggested even the two proteins possess similar three-dimensional structure, the zinc-ion binding alter the contact network among residues, further change the folding mechanism. The methods should be common mechanism for metal ions cofactor to modulate the folding process.Study three:In addition to non-covalent binding cofactor, phosphate group can be regard as covalent binding cofactor for phosphorylation modification protein af-ter translation. The phosphorylation can alter the electrostatic character and local physical-chemistry environment to modulate conformation and dynamics of protein, play an important role in cell signal transduction. For modulating by phosphorylation, little is known about conformation and dynamics. In this work, a short peptide in-clude serine/threonine as study models, we investigated the molecular mechanism of backbone conformational propensity by phosphorylation modulate with all-atom repli-ca exchange molecular dynamics simulation. The results showed that phosphorylation can increase backbone group hydration of serine, so increase the propensity of PPII; for threonine, the phosphorylation decrease backbone group hydration, and decrease the propensity of PPII, but a stronger hydrogen-bond can form between side-chain phos-phate and backbone NH group, the interaction similar to geometric constraint in favor of helix conformation. These results showed that phosphorylation can make obvious-ly effects for backbone conformational propensity of serine and threonine, and enrich the knowledge of phosphorylation, have important value for research of intrinsic dis-ordered protein.The research results is of significance to understand the effects of cofactor on pro-tein folding and conformational distribution. The contents in the thesis are arranged below:In chapter one, we introduce the background knowledge of protein conforma-tion, folding, cofactor as well as simulation methods. In chapter two, we introduce the work about the effects of heme-binding on myoglobin folding by structure-based coarse-grained simulation. In chapter three, we introduce the work about the difference of folding mechanisms for homologous protein are modulated by structural Zn(Ⅱ). In chapter four, we will introduce the work about the effects of phosphorylation on intrin-sic propensity of short peptide. Finally, in chapter five, we will give conclusions about the thesis and give suggestion for further study. |
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