The Development And Applications Of Metalloprotein Force Field

There are nearly one third proteins contain metal ions in the world. The metal ions play cardinal role in both the structure and function of proteins in most of life processes. Metalloproteins serve crucial functions in cell, such as enzyme catalysis, transport and storage of proteins, signal transmissions. For detail example, the calcium binding process of calcium contained protein named calbindin is the central process in signal transmission in cell, copper-contained protein, cuprein relates intimately to the redox reactions in living body, and iron-contained protein, ferritin, such as myoglobin and hemoglobin can transmute and store oxygen. As a member of zinc-contained protein, zinc finger protein, zinc finger, involves to various biology functions, such as DNA recognition, resembling of RNA, transcription, and regulation of kinase. In current research, the study of macromolecules in biology, such as proteins, is mainly limited into area of classi-cal mechanics. The general molecular force fields, such as AMBER, CHARMM, GROMOS, OPLS, etc. obtained triumph in the simulations of proteins, nucleic acids, and saccharides. While till today the theoretical research of metalloprotein is still in the initial state. The more developed model is bonded model, in which the metal ions explicitly bonded to ligating atoms. But in the active site of cat-alytic metalloproteins, the coordinate number of metal ion may change according to environment. In this case, the non-bonded model is demanding. Alas, in the popular molecular force fields, the use of non-bonded model is limited. So, the study of molecular dynamics (MD) modeling metalloprotein is of profound mean-ing for help understand the biophysical properties of metalloproteins, predict and design novel metalloproteins. For precisely describe the metal ions in metalloproteins, this thesis aims to novel charge scheme for metalloprotein, based upon a kind of partial charge devised from our group, named protein-specific polarized charge (PPC). We utilize and advance the thought of PPC, calculate the metal-binding site using quantum chemistry, then fit the partial charges of the metal ions and its first ligating shell, with the calculated polarized charge density. The organization of this thesis is like this:first we study the viability of existed functionals in modeling metal system, and then we study how the enhanced sampling technique helps to refine the results of MD simulations. At last, with the experience from modeling, we devise the PPC charges for metalloprotein and apply this MD simulation scheme into two metalloproteins, DFsc and DF1.Based on that thought, in chapter three, we study an active de novel de-signed protein, DFsc. It contains two metal ions. The ions can be iron, zinc, manganese, chromium, etc. While only the iron or zinc coordinated DFsc is with activity to catalyze oxidation reaction. In this protein, there are two neighboring zinc ions posited in the bottom of a solvent-accessible channel. The geometry of the zinc-binding group is asymmetric. Furthermore, the coordinate numbers of the two zinc ions are also changeable. We study this system using MD simulation with PPC charge, and update the charges of zinc binding group on-the-fly (adap-tive PPC, APPC). Meanwhile, four AMBER force fields (AMBER94, AMBER99, AMBER99SB, and AMBER03) are used as control group. Firstly, from RMSD analysis we find the backbone of DFsc is stabilized during MD simulation under APPC. And the NMR violations data taken from MD trajectory indicates the highly accordance to experimental value. Both of the results validate the simu-lation of backbone is correct. Then, from the analysis of both the time-evolution plots of bond length and their averaged values indicate one of the zinc ions is four-fold coordinated and the other is five-fold coordinated, so the asymmetry of the zinc binding group has been maintained. And the trajectories reveal there can be great fluctuation of the zinc ions even after the backbone has reached its equilibrium. According previous works in other group, we assign this fluctuation to the activity of this zinc binding group. At last, after the analysis of the cross correlation between water molecule around the zinc ions and the zinc-zinc dis-tance, we have found the cause of the fluctuation is the perturbation of water to zinc ions. On the contrast, in all the trajectories from AMBER force fields, the zinc ions are over stable and the coordinate numbers are all incorrect.While, whether the fluctuation of zinc ions initiates from the character of the DFsc or from our MD simulation strategy is not clear in the single case of DFsc. So in the chapter four, we have simulated an inactive analogue of DFsc, DF1. In DF1, the solvent accessible channel is blocked by mutant residues. So the zinc ions are buried in the protein and can never interact to solvent. The simulated results of DF1 indicate the zinc-zinc distance is much stable during the MD run, and its value is stabilized around 3.74A. So this evidence approves the charge scheme used here can reflect the character of metal ions in proteins correctly. And the AMBER force field always overestimates the stability of zinc ions.Currently, density functional theory (DFT) is the most popular calculation method in quantum chemistry. But for metal-contained system, there is no elixir which can be used in every system. For specific system, one must test the viability of the combination of functional and basis. In chapter five, we have thoroughly studied the influence from different functional to the geometry and UV-vis spec-tra of ferrocene. The results indicate the calculated geometries of these com-pounds are nearly the same under different functional, although their UV-vis spectra change much. We found the combination PBEO/6-311++G** with po-larizable continuum solvation model (PCM) can give the most accurate UV-vis spectra. And we also successfully apply this combination to the UV-vis spectra calculations of other metal-contained systems, including bis-(benzene) chromium and four of derivatives of ferrocene, namely, acetylferrocene (AcFc), hydroxyethyl-ferroene (HyFc), vinylferrocene (ViFc), and ethynylferrocene (EtFc).Sampling is an important step of MD simulation. Via correct and highly efficient sampling, one can obtain much accurate free energy. This is the central task of the studying of protein-ligand binding. To test whether the application of enhanced sampling technique under conventional force field can obtain sufficiently accurate free energy, in chapter six, we have studied relative binding free energy,△△G, between the binding of protein Avidin and charged ligand biotin and other complex, BTN2/avidin, using the high accuracy sampling technique, thermody-namic integration (TI), under classical AMBER force field. We find although the trajectories are stable, and the simulation time is long enough, the calculated△△G is still under estimated comparing with the experimental value (calculated value is Ca. lkcal/mol, while the experimental value is 6kcal/mol). This re-sult is much similar to the previous value produced by P. A. Kollman et al. in which the△△G was calculated by MM-PBSA method under AMBER force field. During the simulation, there are four of hydrogen bonds had changed. In two of them, ligand serves as donor, and in the other two, ligand serves as acceptor. This may cause the cancellation of the△△G, which is generated mainly from the hydrogen-bond change between the two complexes. This result also proves that there may be large deviation when doing simulations of charged system under the non-polarizable classical force field.