We designed and implemented a suite of program which is able to accurately and automatically predict chemical shift of protein C-alpha nuclei on the simple basis of protein sequence and low-resolution C-alpha trace conformation. We applied this knowledge-based prediction approach on a group of C-alpha structures generated by computational modeling methods, and successfully identify the native structure by comparing the predicted and unassigned observed NMR data. We begin the automatic prediction with construction of a knowledge-based protein structural profile library, which aims at capturing the most significant structural features affecting chemical shifts, even from a highly coarse-grained C-alpha model. The library is populated by more than 5000 non-homologous proteins, with publicly accessible structures from Protein Data Bank and more than 1.5 million pre-calculated chemical shifts by a widely used NMR predictive program SHIFTX. Fed with the minimum sequential and structural information, the program is able predict highly consistent chemical shifts comparing with experimental observed data from an NMR spectroscopy database BioMagResBank(BMRB). Overall, the proposed program achieves a correlation coefficient of 0.937 and RMSD of 1.702 ppm towards observed chemical shifts. These results are slightly lower than those from achieved by the benchmark program SHIFTX, which utilizes semi-empirical hypersurfaces and semi-classical equations. On the same test sets, SHIFTX achieved a correlation coefficient of 0.945 and RMSD of 1.599 against experimental observations. In compensation, like most other predictive methods, SHIFTX requires high-resolution protein structures with three-dimensional all-atom coordinates, its accuracy of prediction will be highly compromised unless fed with all-atom high-resolution structure, which is normally exceedingly difficult to obtain. Combined with an optimization matching system using Monte Carlo method, we compared the predicted C-alpha chemical shifts with unassigned NMR data from BMRB, and successfully identify the native fold topology by the resemblance between two sets of chemical shifts. In summary, the proposed program is one of the only methods which are capable to predict accurate chemical shifts, even on low-resolution C-alpha protein structures, which are far more accessible and readily obtained by currently available protein modeling methods. Based on the understanding that the similar pattern of chemical shifts reflects resemblance of two structures, we approved that prediction-recognition approach not only fundamentally improve the way of the NMR-assisted computational protein modeling, but is effective in accelerating the traditional protein structure determination and validation by NMR.

Lipid bilayer membranes are ubiquitous in biology and electrostatics play a key role in their functionality. The interfacial electrostatics of lipid bilayers involves interplay between the surface potential and charge regulation in the form of ion binding, protonation and lipid mobility. Mobile lipid charge regulation in particular is unique to lipid interfaces and is thought to be an important factor in charged macromolecule-membrane interactions. We used Atomic Force Microscopy (AFM) for the first nanometer scale experimental demonstration of mobile lipid charge regulation occurring in supported lipid bilayer membranes. By combining finite element computer simulations and experimental AFM data, we showed that mobile lipid charge regulation accounts for the short range deviations from the expected electrostatics over anionic lipids. We also accounted for van der Waal interactions and electrolyte ion binding in our calculations and found the mobility of the lipid to be the dominant factor in the short range deviations. Control experiments on silicon nitride surfaces, whose surface charges are immobile, showed that the short range deviation could be accounted for by the formation of a stem layer due to cation binding. Further evidence for tip-induced mobile lipid charge regulation was presented in the form of clear differences in the short range electrostatics of mobile fluid phase lipids when compared to immobile gel phase lipids. Furthermore, our data confirmed the theoretically predicted differences between surfaces containing mobile versus immobile charges.

<p>We study the comparison of computational simulations of biomolecules to experimental data. We study the convergence of these simulations to equilibrium and determine measures of variance of the data using statistical methods. We run replica-exchange molecular dynamics (REMD) simulations of eight helical peptides and compare the simulation helicity to the experimentally measured helicity of the peptides. We use one-way sensitivity analysis to determine which parameter changes have a large effect on helicity measurements and use Bayesian updating for a parameter of the AMBER potential. We then consider the theoretical convergence behavior of the REMD algorithm itself by evaluating the properties of the isothermal numerical integrators used in the underlying MD. The underlying constant-temperature integrators explored in this thesis represent a majority of the deterministic isothermal methods used with REMD simulations and we show that these methods either fail to be measure-invariant or are not ergodic. For each of the non-ergodic integrators we show that REMD fails to be ergodic when run with the integrator. We give computational results from examples to demonstrate the practical implications of non-ergodicity and describe hybrid Monte Carlo, a method that leads to ergodicity. Finally, we consider the use of stochastic Langevin dynamics to simulate isothermal MD. We show geometric ergodicity of the Langevin diffusion over a simplified system with the eventual goal of determining geometric ergodicity for Langevin dynamics over the full AMBER potential.</p> / Dissertation

Thesis (Ph.D.)--Lehigh University, 2009. / Adviser: James D. Gunton.

Thesis (Ph. D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3364. Adviser: David A. Agard.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3637. Adviser: K. D. Bhalerao. Includes bibliographical references (leaves 69-78) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2701. Adviser: Eric Jakobsson. Includes bibliographical references (leaves 120-147) Available on microfilm from Pro Quest Information and Learning.

<p> In this thesis, we discuss three topics concerning extracellular electron transfer in the Dissimilatory Metal Reducing Bacterium (DMRB) <i>Shewanella oneidensis</i> MR-1. One proposed strategy to accomplish extracellular charge transfer in <i>Shewanella</i> involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as bacterial nanowires within biofilms. In the first part of this dissertation, to describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium <i>S. oneidensis</i> MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms. </p><p> In the second part, we report the first <i>in vivo</i> observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe <i>S. oneidensis</i> MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to <i> S. oneidensis</i> MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by our proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. </p><p> In addition, to elucidate the membranous nature of <i>Shewanella </i> nanowires, we imaged these filaments using Transmission Electron Microscopy. The TEM images reported in this thesis also provide the most accurate estimates of bacterial nanowire dimensions to date. Future TEM and cryo-TEM imaging can establish the specific alignment and configuration of outer membrane cytochromes that facilitate electron transport along bacterial nanowires. </p><p> In the third part of this thesis, we focus on the molecular conductance of MtrF, the first decaheme outer membrane cytochrome with a solved crystal structure. Decaheme outer membrane cytochromes of <i>Shewanella</i> play a crucial role in all the suggested pathways of extracellular electron transfer. An understanding of the electron transfer properties in MtrF will therefore impact all aspects of extracellular electron transfer research. In this thesis, using purified MtrF, we form monolayers of the protein on atomically flat gold substrates and address the dry monolayer with a Scanning Tunneling Microscope (STM) tip. This technique can be used in the future to examine the conductivity of individual MtrF molecules within the monolayer in the form of I-V curves. This methodology will allow experimental comparison with recently developed simulations of MtrF conductance.</p>

Twenty years ago the experiments of Hotani revealed that flagellar polymorphism (the ability of bacterial flagellar filaments to take on different quaternary structures, specifically helices of different handedness and pitch) can be generated by fluid stresses of the same magnitude as those that occur during natural swimming. Experimental work including the recent crystallization of flagellin, as well as theoretical studies, show how the packing properties and underlying bistability of flagellin may give rise to different static structures. Hotani's experiments showed dynamic nucleation and propagation of domains of opposing handedness on a single flagellum. Here we present the first theory to explain this phenomenon, which is of great relevance to the study of the bundling-unbundling transition in run-and-tumble behaviour of free-swimming bacteria. Our model, based entirely on measurable, physical properties of flagella, bridges the gap between protein-scale statics and cell-scale dynamics. We generate simulations of flagellar motion under fluid stress that exhibit nucleation rates and transition speeds in quantitative agreement with experiment.

big brain is a neurogenic gene which, when mutated causes defects in cell fate determination during Drosophila neurogenesis through an unknown mechanism. The protein Big Brain (BIB) has sequence identity with the Major Intrinsic Protein family including the water- and ion-conducting Aquaporin channels. We show here that BIB expressed heterologously in Xenopus oocytes is a non-selective monovalent cation channel with permeability to K⁺ > Na⁺ >> TEA⁺. BIB macroscopic conductance, activated in response to endogenous oocyte signaling pathways, was decreased after treatment with 20μM insulin and was enhanced with 10μM lavendustin A, a tyrosine kinase inhibitor. Current activation is not observed in control oocytes or in oocytes expressing a non-functional mutant BIB channel (E71N) that is expressed on the plasma membrane, as confirmed with confocal microscopy and western blotting. Cell-attached patch clamp experiments revealed a novel large conductance (300 ± 30pS) channel in BIB-expressing but not control oocytes. Divalent cations, such as calcium, are important developmental signaling molecules. We found that calcium and barium partially block currents in BIB-expressing oocytes. We further demonstrated that a conserved glutamate (E71) located in transmembrane domain 1 is crucial for channel properties of BIB. Mg²⁺ block was introduced in currents from oocytes expressing the BIB mutant E71D. The carboxy tail of BIB comprises 61% of the channel (431 of 700 residues) and contains sites of potential serine/threonine and tyrosine phosphorylation, SH3 binding domains, PDZ binding domains and three polyglutamine stretches. The importance of the carboxy tail for BIB channel activity was demonstrated by truncation of the channel at two sites. Truncated channels had reduced whole-cell conductance and at least one (Δ317) was not tyrosine phosphorylated. In summary, the results presented in this dissertation provide a novel function of the Drosophila protein Big Brain as a regulated cationic channel, indicate that BIB can participate in tyrosine kinase-regulated transmembrane signaling, and suggest a role for membrane depolarization in the neurogenic function of BIB in early development.