<p>Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.</p><p>This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.</p><p>The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases. </p><p>To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.</p> / Dissertation

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis. Using laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin g (IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis. Here we have provided evidence that membrane rafts serve as platforms that are used to pre-cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation, followed by internalization to the Golgi complex.

<p>Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.</p><p> In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.</p><p> </p><p> We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.</p><p> To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.</p><p> The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.</p> / Dissertation

No description available.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 271 p.; also includes graphics. Includes bibliographical references (p. 245-269). Available online via OhioLINK's ETD Center

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This assay format comprises the displacement of receptor molecules bond to immobilized ligand by a competition reaction with ligand in solution. This is beneficial because displacement of high molecular receptors is detected compared to low molecular ligand as in classical binding analysis therefore potentially lowering the method detection limit. It was hypothesized that with choosing appropriate measuring methods and theoretical modeling reaction rate constants can be determined separately in every kinetic stage of the assay format. Herein elucidating the dominant valence of antibody antigen binding in the established assay was of great importance. Using the Influenza Hemagglutinin (HA) peptid binding to mono or bivalent Anti-Hemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and Anti-Hemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibody. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding; this proves the quality of SPR measurements and demonstrates consistence between measurement, simulation, and binding model. Maximum decline of SPR response could be used to determine ligand concentrations in analyte. Displacement dependence from antigen concentration was found to be exponential and was explained by rebinding. Kinetic data and models could be transferred for the simulation of almost stationary displacement assay formats realized with impedance and fluorescence spectroscopy. With the obtained results it was possible to detect the displacement of the bacterial signaling autoinducer AI-2 by a displacement assay format using periplasmic binding protein LuxP as receptor. Concluding it can be said that the hypothesis could be proved and the obtained results can facilitate the use of displacement assay formats in biosensing. Displacement assay formats should be especially interesting in small molecule detection and in compact integrated mass sensitive sensor designs suitable as mobile sensors in outdoor screening. / Die Analyse des Bindungsverhaltens niedermolekularer Liganden an Proteine ist für die Aufklärung von biologischen Regulationssystemen oder bei der Suche neuer medizinischer Wirkstoffe von Wichtigkeit. Ein markierungsfreies Detektions¬prinzip zur Erfassung niedermolekularer Liganden ist die Displacement- oder Replacement-Methode. Bei dieser tritt die Bindung des Rezeptors an den immobilisierten Liganden mit der Bindung an freien Liganden in Konkurrenz, sodass anstelle der niedermolekularen Liganden die hochmolekularen Rezeptoren detektiert werden können. In dieser Arbeit wurde von der Hypothese ausgegangen, dass durch die Auswahl geeigneter Messverfahren und der zugeordneten Modellierung die einzelnen kinetischen Stadien des Displacements separat zur Bestimmung der kinetischen Konstanten der Displacementprozesse genutzt werden können. Dabei sollte unter anderem auch eine Aussage über die dominierende Valenz der Antigen-Antikörper-Bindung erreicht werden. Hierzu wurden auf der Basis des Modellsystems Hämagglutinin-Peptid/ Hämagglutinin-Antikörper Displacement-Assays mit mono- und bivalenten Anti-körpern entwickelt, anhand derer eine genaue zeitaufgelöste Analyse des Bindungs- und Ablösungsverhaltens vom Liganden HA an den Anti-HA-Antikörper (Rezeptor) mittels Oberflächenplasmonenresonanz(SPR)-Spektroskopie erzielt wurde. Ausgehend von den Reaktionsgleichungen zwischen Liganden und mono- und bivalenten Rezeptoren wurden mathematische Modelle entwickelt, die eine exakte Simulation der SPR-Ergebnisse ermöglichten. Durch genaues Anpassen der Simulationsplots an die Messplots konnten alle Ratenkonstanten, die die Kinetik der Reaktionen zwischen Liganden, Rezeptoren und ihren Komplexen bestimmen, ermittelt werden. Da auch für eine große Variation der Rezeptorkonzentrationen in der Analytlösung nahezu identische Werte für die Ratenkonstanten erhalten wurden, ergeben Messungen und Simulationen ein konsistentes Bild der Anbindungskinetik und bestätigen die Qualität der Messungen. Aus Messungen des maximalen Responsabfalles kann die Konzentration der freien Antigene beim Displacement ermittelt werden. Man findet eine exponentielle Abhängigkeit des Displacements von der Konzentration der freien Antigene, die sich durch den sogenannten „Rebindingeffekt“ erklären lässt. Die gewonnenen kinetischen Daten und entwickelten Modellierungsverfahren konnten zur Simulation quasistationärer Detektionsverfahren, die mit Fluoreszenz- und Impedanzspektroskopie durchgeführt wurden, erfolgreich angewandt werden. Die erzielten Erkenntnisse konnten auf ein wissenschaftlich herausforderndes biologisches System (LuxP/AI2) angewandt werden, bei dem das niedermolekulare Signalmolekül AI2 über ein Displacementassay detektiert wurde. Dieses System ermöglicht einen Einblick in die Intra- und Interspezieskommunikation bei Bakterien. Insgesamt zeigt sich, dass die hier formulierte Hypothese als bewiesen angesehen werden kann. Die in dieser Arbeit gewonnenen Erkenntnisse eröffnen verschiedene Einsätze der Displacementmethode in der Biosensorik. Insbesondere lassen sich damit kleine Moleküle markierungsfrei quantitativ bestimmen, ohne hoch präzise Analysengeräte einsetzen zu müssen. Damit ergibt sich die Möglichkeit, sehr kompakte integrierte massensensitive Sensoren, die nicht die Empfindlichkeit hochempfindlicher SPR-Spektrometer erreichen, zur Detektion kleiner Moleküle einzusetzen. Dies ist besonders für mobile Anwendungen von Interesse.

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This assay format comprises the displacement of receptor molecules bond to immobilized ligand by a competition reaction with ligand in solution. This is beneficial because displacement of high molecular receptors is detected compared to low molecular ligand as in classical binding analysis therefore potentially lowering the method detection limit. It was hypothesized that with choosing appropriate measuring methods and theoretical modeling reaction rate constants can be determined separately in every kinetic stage of the assay format. Herein elucidating the dominant valence of antibody antigen binding in the established assay was of great importance. Using the Influenza Hemagglutinin (HA) peptid binding to mono or bivalent Anti-Hemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and Anti-Hemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibody. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding; this proves the quality of SPR measurements and demonstrates consistence between measurement, simulation, and binding model. Maximum decline of SPR response could be used to determine ligand concentrations in analyte. Displacement dependence from antigen concentration was found to be exponential and was explained by rebinding. Kinetic data and models could be transferred for the simulation of almost stationary displacement assay formats realized with impedance and fluorescence spectroscopy. With the obtained results it was possible to detect the displacement of the bacterial signaling autoinducer AI-2 by a displacement assay format using periplasmic binding protein LuxP as receptor. Concluding it can be said that the hypothesis could be proved and the obtained results can facilitate the use of displacement assay formats in biosensing. Displacement assay formats should be especially interesting in small molecule detection and in compact integrated mass sensitive sensor designs suitable as mobile sensors in outdoor screening.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 157 / Die Analyse des Bindungsverhaltens niedermolekularer Liganden an Proteine ist für die Aufklärung von biologischen Regulationssystemen oder bei der Suche neuer medizinischer Wirkstoffe von Wichtigkeit. Ein markierungsfreies Detektions¬prinzip zur Erfassung niedermolekularer Liganden ist die Displacement- oder Replacement-Methode. Bei dieser tritt die Bindung des Rezeptors an den immobilisierten Liganden mit der Bindung an freien Liganden in Konkurrenz, sodass anstelle der niedermolekularen Liganden die hochmolekularen Rezeptoren detektiert werden können. In dieser Arbeit wurde von der Hypothese ausgegangen, dass durch die Auswahl geeigneter Messverfahren und der zugeordneten Modellierung die einzelnen kinetischen Stadien des Displacements separat zur Bestimmung der kinetischen Konstanten der Displacementprozesse genutzt werden können. Dabei sollte unter anderem auch eine Aussage über die dominierende Valenz der Antigen-Antikörper-Bindung erreicht werden. Hierzu wurden auf der Basis des Modellsystems Hämagglutinin-Peptid/ Hämagglutinin-Antikörper Displacement-Assays mit mono- und bivalenten Anti-körpern entwickelt, anhand derer eine genaue zeitaufgelöste Analyse des Bindungs- und Ablösungsverhaltens vom Liganden HA an den Anti-HA-Antikörper (Rezeptor) mittels Oberflächenplasmonenresonanz(SPR)-Spektroskopie erzielt wurde. Ausgehend von den Reaktionsgleichungen zwischen Liganden und mono- und bivalenten Rezeptoren wurden mathematische Modelle entwickelt, die eine exakte Simulation der SPR-Ergebnisse ermöglichten. Durch genaues Anpassen der Simulationsplots an die Messplots konnten alle Ratenkonstanten, die die Kinetik der Reaktionen zwischen Liganden, Rezeptoren und ihren Komplexen bestimmen, ermittelt werden. Da auch für eine große Variation der Rezeptorkonzentrationen in der Analytlösung nahezu identische Werte für die Ratenkonstanten erhalten wurden, ergeben Messungen und Simulationen ein konsistentes Bild der Anbindungskinetik und bestätigen die Qualität der Messungen. Aus Messungen des maximalen Responsabfalles kann die Konzentration der freien Antigene beim Displacement ermittelt werden. Man findet eine exponentielle Abhängigkeit des Displacements von der Konzentration der freien Antigene, die sich durch den sogenannten „Rebindingeffekt“ erklären lässt. Die gewonnenen kinetischen Daten und entwickelten Modellierungsverfahren konnten zur Simulation quasistationärer Detektionsverfahren, die mit Fluoreszenz- und Impedanzspektroskopie durchgeführt wurden, erfolgreich angewandt werden. Die erzielten Erkenntnisse konnten auf ein wissenschaftlich herausforderndes biologisches System (LuxP/AI2) angewandt werden, bei dem das niedermolekulare Signalmolekül AI2 über ein Displacementassay detektiert wurde. Dieses System ermöglicht einen Einblick in die Intra- und Interspezieskommunikation bei Bakterien. Insgesamt zeigt sich, dass die hier formulierte Hypothese als bewiesen angesehen werden kann. Die in dieser Arbeit gewonnenen Erkenntnisse eröffnen verschiedene Einsätze der Displacementmethode in der Biosensorik. Insbesondere lassen sich damit kleine Moleküle markierungsfrei quantitativ bestimmen, ohne hoch präzise Analysengeräte einsetzen zu müssen. Damit ergibt sich die Möglichkeit, sehr kompakte integrierte massensensitive Sensoren, die nicht die Empfindlichkeit hochempfindlicher SPR-Spektrometer erreichen, zur Detektion kleiner Moleküle einzusetzen. Dies ist besonders für mobile Anwendungen von Interesse.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 157

<p> According to the World Health Organization, long-term exposures to heavy metal toxicants such as arsenic (As) and lead (Pb), even at the parts per billion (ppb, μg/L) level, can cause severe health problems such as cancer, skin lesions, and cardiovascular diseases. Therefore, an accurate and rapid heavy metal toxicant monitoring technique is needed. This research investigated the proof-of-the concept of a portable sensor for detecting As and Pb in water. The sensor system utilized a Quartz Crystal Microbalance - QCM (openQCM w-i2) system interfaced with a computer as the sensing platform. It was further integrated with a peristaltic pump and required tubing to create the integrated sensing system. It used a 10 MHz AT-cut quartz crystal gold electrode as the sensing substrate. For the determination of As in water, dithiothreitol (DTT) was used as the ligand to be deposited on the gold electrode using the Self-assembly-monolayer method (SAM). For the determination of Pb, a combination of ligands (Chitosan, Glutaraldehyde, and lead ionophore II - CGL) was used and deposited on the gold electrode using the spin-coating method. The system was tested for As in water with specific concentrations (0, 50, 100, and 200 ppb) under laboratory conditions. Similarly, the system was tested for Pb in water with different concentrations (0, 10, 25, 50, and 100 ppb) under laboratory conditions. The resulted change of frequency (with respect to time, in seconds) of the QCM system to different concentrations of the individual analyte was recorded. Subsequently, the recorded data were analyzed to determine the correlation model and coefficient of determination, R2. The maximum R2 values for detecting As and Pb were 0.963 and 0.991, respectively. Thus, this proof-of-the-concept study using the developed QCM-based sensing system for detecting As and Pb in water was successful.</p>

<p>Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.</p><p>The thermodynamic integration technique is used to calculate p<i>K</i>_a values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated p<i>K</i>_a values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine p<i>K</i>_a shifts in wild-type and mutant epoxide hydrolase. The calculated p<i>K</i>_a values support a model that can explain some of the pH dependent properties of this enzyme.</p><p> Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.</p><p>Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.</p>

ATP-Binding-Cassette (ABC) transporters are primary active pumps that typically couple the binding and hydrolysis of ATP to the translocation of compounds across cellular membranes. Some, like ABCB1, ABCC1 and ABCC3, are polyspecific and can efflux clinically important drugs which may contribute to their therapeutic failure. In this study I have investigated (1) the mechanism of ABC transporter function, (2) studied the potential for regulation by ubiquitin ligases (both using ABCB1 as a model), and (3) tested the involvement of ABCC1 and ABCC3 in autocrine signalling in cancer. (1) In 1966, Jardetzky et. al [1] proposed that membrane pumps function by exposing their ligand-binding pocket alternately on different sides of the membrane. For ABC transporters, this coupling of the aspect and affinity of the ligand-binding cavities of the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism but is poorly understood at the molecular level. Structure data suggest signals are transduced through intracellular loops of the TMDs which slot into grooves on the top surface of the NBDs. At the base of these grooves is the Q-loop. By analysing the function of Q-loop mutants in combination with ligand binding cavity mutants I have discovered that the Q-loops are crucial to the transport cycle and that they are required to couple ligand binding to conformational changes at the NBDs necessary to drive the transporter into an inward closed state. 4 (2) ABCB1 is known to be a key component of chemical barrier separating the circulation from the cerebrospinal fluid. It has also been reported to transport β-amyloid across the lumenal membrane and into the circulation. Loss of ABCB1 from the barrier with age has therefore been suggested to play a role in Alzheimer’s Disease. The ubiquitin ligase Nedd4-1 has been implicated in the post-translational regulation of ABCB1 abundance in cells. Here, I report that ABCB1 can be ubiquitinated by Nedd4-1 in vitro and identify the residues modified (by mass spectrometry). (3) Lysophosphatidylinositol (LPI) is an autocrine metabolite produced by cancer cells that binds to the G-protein coupled transmembrane receptor GPR55 on the surface of cells. Stimulation of GPR55 activates a signalling cascade that induces proliferation and metastases of the cancer cells. How LPI is released from the cells was not known. In this study I show that ABCC1 and ABCC3, which are known to be expressed in ovarian and pancreatic cancers, can transport LPI into inside-out vesicles suggesting a new role for these “drug resistance” transporters in cancer biology.