Research On The Mechanism For Electron Transfer Along Microbial Nanowires

Microbial extracellular respiration is a process of anaerobic metabolism,which transfers electrons across cell membranes,from cytoplasm to periplasm and extracellular insoluble acceptors.The electron-transport chain is composed by proteins and small molecules,including c-type cytochromes,nanowires,flavins and quinones.Microbes might choose different pathways of electron transfer under different conditions.Among these pathways,the nanowire of Geobacter sulfurreducens plays an important role during the process of electron transfer,and is of environmental and practical significance because of its metallic-like electrical conductivity and potential in biological energy and bioremediation of pollution.Although the conductivity of the nanowires,which are protein polymers called Type IV Pili,is attributed to the overlap of pi orbitals inside the proteins,there is only a rudimentary understanding of the mechanism for electron transfer along these pili.Because of the flexibility of the Geobacter sulfurreducens Type IV Pili,it is hard to obtain the structural details of them by traditional methods such as X-ray crystallography or cryo-EM.Thus,in this dissertation,bioinformatics methods such as sequence analysis,protein docking and molecular dynamics simulation and bioelectronics experiments such as gene mutation and microbial fuel cell are employed to explore the details of Geobacter sulfurreducens Type IV Pili.These analyses will help us to better understand the special mechanism for electron transfer,and provide evidence and theoretical guides for genetically altering these microbes and improving the efficiency of their electron transfer.Also,our work provides a framework for the researches of microbial nanowires,which will be an efficient and self-improved tool for understanding and explaining the structures and functions of these pili polymers.Detailed contributions of this work can be summarized as four parts.Ⅰ.A method of modeling quaternary structures of microbial type IV pili was developed.Type IV Pili(T4P)and Type II Secretion System(T2SS)pseudopili are filament extending to the outside of microbes,which is homologously composed of subunits called ‘pilins’ in a helically symmetric way.We presented here a new approach to predict pseudo atomic models of pili,combining ambiguous symmetric constraints with sparse distance information obtained from experiments.The method was verified by reconstructing the gonococcal(GC)pilus from Neisseria gonorrhoeae,the type IVb toxin-coregulated pilus(TCP)from Vibrio cholerae,and pseudopilus of the pullulanase T2SS(the PulG pilus)from Klebsiella oxytoca.According to the analyses of computational errors,the subunits should be treated cautiously because of their flexibility.The results of modeling also implied that a pilus might have more than one but fewer than many possible intact conformations.Ⅱ.A model of the conductive pili from Geobacter sulfurreducens was built.In order to determine if it was feasible for the pilin monomers of G.sulfurreducens to assemble into a conductive filament,theoretical energy-minimized models of Geobacter pili were constructed with the approach described in last chapter,in which pilin monomers are assembled using randomized structural parameters and distance constraints.The lowest energy models from a specific group of predicted structures lacked a central channel,in contrast to previously existing pili models.In these models,the three N-terminal aromatic residues of the pilin monomer are arranged in a potentially electrically conductive geometry,sufficiently close to account for the experimentally observed metallic like conductivity of the pili that has been attributed to overlapping pi-pi orbitals of aromatic amino acids.Ⅲ.The conformation of Geobacter sulfurreducens pilin monomer PilA was analyzed under different conditions.All the previous models of G.sulfurreducens pili are based on the assumption that there are few differences between the conformations of PilA in nuclear magnetic resonance experiment and in assembled pili.However,the difference between the experimental and the native assembly environment have never drawn attention.Here,the pilin monomer PilA was investigated by molecular dynamics simulations under three different conditions: in detergent micelles mimicking the NMR experimental environment where it was determined;in a lipid bilayer similar to the inner membrane of Gram-negative bacteria;and an explicit water environment.The results showed significant differences of the pilin conformations from the three different systems,which not only yielded agreement between experimental and simulative data,but also suggested unknown factors for the intactness and functioning of PilA.Ⅳ.The influence of charged amino acid residues in the pilin was analyzed.To better understand the influence of charged residues in the pilin monomer PilA,and verify the theoretical models previously described,several strains of Geobecter sulfurreducens was constructed,in which the targeted charged residues were mutated and the charges of these residues were reversed.These mutants were incubated in microbial fuel cells and the current values of the cells were utilized to decide if the pili function was changed.The experimental results showed that some residues,including Asp39,Arg41,Lys44,Asp53 and Asp54,were important to the Geobacter pili,because the charge reversal of these residues inhibited the electron transfer.Such inhibitions might be attributed to obstruction of interactions between the subunits and the unknown factors or the instability of salt bridges in the assembled pili.These experiments verified the results of the pili modeling and the molecular dynamics simulations of the pilin monomer.