Calmodulin (CaM) is an essential protein found in all eukaryotes ranging from vertebrates to unicellular organisms such as Paramecia. CaM is a calcium sensor protein composed of two domains (N and C) responsible for the regulation of numerous calcium-mediated signaling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. Since the discovery of CaM, most studies have focused on the role of its calcium-saturated form. However, an increasing number of target proteins have been discovered that preferentially bind apo (calcium-depleted) CaM. My study focused on understanding how apo CaM recognizes drugs and protein sequences, and how those interactions differ from those of calcium-saturated CaM. I have used spectroscopic methods to explore CaM binding the drug Trifluoperazine (TFP) and the IQ-motif of the type 2 Voltage-Dependent Sodium Channel (Nav1.2IQp). These studies have shown that both TFP and Nav1.2IQp preferentially bind to the "semi-open" conformation of apo CaM. TFP was shown to be an unusual allosteric effector of calcium binding to CaM. Using 15N-HSQC NMR spectroscopy, I determined the stoichiometry of TFP binding to apo Cam to be 2:1 and to (Ca2+)4-CaM to be 4:1 TFP:CaM. That difference in stoichiometry determined whether TFP decreased or increased the affinity of CaM for calcium. Analysis of residue-specific chemical shift differences indicated that TFP binding to apo and (Ca2+)4-CaM perturbed the C-domain more than the N-domain, prompting high-resolution structural studies of the isolated C-domain of CaM. Crystallographic studies of TFP bound to a calcium-saturated C-domain fragment of CaM (CaM76-148) revealed that CaM adopted an "open" tertiary conformation. The unit cell contained two protein and 4 drug molecules. The orientation of TFP revealed that its trifluoromethyl group was found in two alternative positions (one in each protein in the unit cell), and that Met 144 acted as a gatekeeper to select the orientation of TFP. In contrast to TFP binding to the "open" conformation of calcium-saturated CaM76-148, my NMR studies showed that TFP bound the "semi-open" conformation of apo CaM76-148. TFP interacted with CaM residues near the perimeter of the hydrophobic pocket, but did not contact residues that are solvent-accessible only in the "open" form. Allosteric effects due to TFP binding were observed in the calcium-binding loops of apo CaM76-148. These properties suggest that TFP may antagonize interactions between apo CaM and target proteins such as ion channels that preferentially bind apo CaM. Nav1.2, is responsible for the passage of Na+ ion across cellular membranes. Apo binding of CaM to Nav1.2 poises it for action upon calcium release in the cell. My NMR studies of CaM binding to the Nav1.2 IQ-motif sequence (Nav1.2IQp) showed that the C-domain of apo CaM was necessary and sufficient for binding. My high-resolution structure of the isolated C-domain of CaM bound to Nav1.2IQp revealed that the domain adopted a "semi-open" conformation. At the interface between the IQ-motif and CaM, the highly conserved I and two Y residues of Nav1.2IQp interacted with hydrophobic residues of CaM, while the invariant Q residue interacted with residues in the loop between helices F and G of CaM. This is the first CaM-IQ complex to be determined by NMR; the only other available structure of apo CaM bound to an IQ-motif was determined crystallographically. To accomplish its regulatory roles in response to cellular Ca2+ fluxes, CaM has evolved multiple binding interfaces that are allosterically linked to its Ca2+-ligation state. My studies of CaM binding to TFP and NaV1.2 demonstrate the versatility of CaM functioning as a regulatory protein comprised of domains having separable functions.

In this work the electrostatics of the pneumatic conveying of granular materials in a non-conducting (PVC) vertical pipe is studied using Electrical Capacitance Tomography (ECT) system. The non-conducting wall in general attains static charges arising from particle-wall collisions in the initial periods of conveying process and then reaches equilibrium with the surroundings. The polarity of particles and conveying pipe inner wall agrees reasonably well with the contact potential difference measurements. The perturbations in the capacitance signal due to charge accumulation are larger with smaller air superficial velocity. The denser flow regimes give larger wall residual charge. Wall charging process shows similar trend by surface potential and ECT measurements. The addition of small amount (0.5% by weight) of anti-static agent (Larostat-519) in the powder form decreases the electrostatic charge generation by altering the patterns for particle-particle and particle-wall collisions. / Singapore-MIT Alliance (SMA)

In this thesis we study the interactions of ions and cationic peptides with a negatively charged lipid bilayer in an ionic solution where the electrostatic interactions are screened. <br /><br /> We first examine the problem of charge renormalization and inversion of a highly charged bilayer with low dielectric constant. To be specific, we consider an asymmetrically charged lipid bilayer, in which only one layer is negatively charged. In particular, we study how dielectric discontinuities and charge correlations among lipid charges and condensed counterions influence the effective charge of the surface. When counterions are monovalent, e. g. , Na⁺, our mean-field approach implies that dielectric discontinuities can enhance counterion condensation. A simple scaling picture shows how the effects of dielectric discontinuities and surface-charge distributions are intertwined: Dielectric discontinuities diminish condensation if the backbone charge is uniformly smeared out while counterions are localized in space; they can, however, enhance condensation when the backbone charge is discrete. In the presence of asymmetric salts such as CaCl₂, we find that the correlation effect, treated at the Gaussian level, is more pronounced when the surface has a lower dielectric constant, inverting the sign of the charge at a smaller value of Ca²⁺ concentration. <br /><br /> In the last chapter we study binding of cationic peptides onto a lipid-bilayer membrane. The peptide not only interacts electrostatically with anionic lipids, rearranging their spatial distributions, but it can also insert hydrophobically into the membrane, expanding the area of its binding layer (i. e. , the outer layer). We examine how peptide charges and peptide insertion (thus area expansion) are intertwined. Our results show that, depending on the bilayer's surface charge density and peptide hydrophobicity, there is an optimal peptide charge yielding the maximum peptide penetration. Our results shed light on the physics behind the activity and selective toxicity of antimicrobial peptides, i. e. , they selectively rupture bacterial membranes while leaving host cells intact.

The organization of two or more biological macromolecules into a functioning assembly is critical for many biological functions to occur. This phenomenon is the result of subtle interplay between complimentary structural and electrostatic factors. While a growing protein data bank of solved protein structures provides experimental evidence for studying the structural factors that stabilize protein-protein interface, there has been little advance in experimental determination of the electrostatic contributions. This lack of experimental investigation into protein electrostatics results in an inability to describe or predict how protein-protein complexes are arranged and stabilized. This problem is addressed in this dissertation by use of vibrational Stark effect (VSE) spectroscopy in which the spectral transitions of a vibrational probe are directly related to the strength and direction of the electric fields in the vicinity of the probe. The work presented here details an approach using VSE spectroscopy coupled with molecular dynamics simulation (MD) to interpret the role that electrostatics play in organizing the signaling protein Ras' interactions with its downstream effectors Raf and Ral guanosine dissociation simulator (RalGDS). Each chapter describes a specific set of experiments and MD simulations designed to understand the nature of protein-protein interactions. In Chapter 3, changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface was found. The areas of conserved Stark shifts are used to help describe electrostatic factors that stabilize the Ras-Ral interface. In Chapter 4, VSE is used to describe an electrostatic origin to the binding tilt between complexes formed between Ras and its two effectors Raf and Ral. There are three regions of conserved Stark effect shifts upon docking with WT Ras between the two effectors, indicating that the docked complexes conserve electrostatic fields, resulting in different binding orientation of otherwise structurally similar proteins. Chapter 5 details the use of MD simulation in correlation with VSE data for 18 mutants of the Ras at the oncogenic position 61 site. The combination of experimental and simulations support the hypothesis that position 61 on Ras is used to coordinate an active site water molecule during native guanosine triphosphate (GTP) hydrolysis. / text

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In recent years, technologies using supercritical water have gained considerable attention, mainly due to versatility and uniqueness of water at elevated temperatures and pressures. The physical conditions required to generate supercritical water also make it prone to large intrinsic thermal and density fluctuations, exacerbated if there are impurities present in the system. These fluctuations induce nucleation, the initial stage of a first-order phase transition, and subsequent mixing of the new phase within the original phase. When this new phase reaches its critical size it grows irreversibly to macroscopic proportions, otherwise, tending to disintegrate. The presence of a polydispersed solid phase within the supercritical phase is responsible for unfavorable phenomena such as particle deposition and corrosion of structural components, both of which result in decreased efficiency and reliability of the supercritical water employing process. Molecular Dynamics (MD) simulation method has been the primary tool of investigation. Molecular motions are tracked on the femto and picosecond time-scales which are particularly important for the study of nucleation. Sodium chloride has been chosen in this research since it is computationally tractable and is unavoidably involved in most industrial water based applications. Cluster size distributions, the size of critical nuclei and cluster life-times are reported. The size distribution of emerging clusters shows a very strong dependence on the system’s density, with larger clusters preferentially formed at lower densities. Also, a materials science application is presented where the rapid quenching of hydrothermally formed sodium chloride clusters leads to a variety of nanostructures, characterizable by prominent vibrational modes. And lastly, during the conditions prior to crystallization, water is not only physically adsorbed to the cluster’s surface but also exists in a “confined” state within subsurface regions for several picoseconds during the nucleation process. A mechanism for the sodium chloride hydrolysis reaction is presented as well as showing that asymmetric electrostatic fields generated by the coalescing ions are on the order of 1010 V/m, sufficient to drive the hydrolysis of confined water molecules. The HCl molecule and hydroxide ions are formed, with the latter segregating preferentially to sub-surface regions in the amorphous NaCl particles. Both HCl and hydroxide are implicated in corrosion. / Thesis (Master, Chemistry) -- Queen's University, 2008-11-03 13:54:56.021

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Thesis (Ph. D.)--University of Sydney, 2007. / Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Pharmacy, Faculty of Pharmacy. Includes bibliographical references. Also issued in print.

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Sherrill, C. David; Committee Member: Bredas, Jean-Luc; Committee Member: El-Sayed, Mostafa A.; Committee Member: Harvey, Stephen C; Committee Member: Hernandez, Rigoberto.

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2009. / Committee Chair: William. Alan Doolittle; Committee Member: Jeffrey Nause; Committee Member: Linda S. Milor; Committee Member: Shyh-Chiang Shen; Committee Member: Stephen E. Ralph.