Noncovalent interactions play a vital role throughout much of chemistry. The understanding and characterization of these interactions is an area where theoretical chemistry can provide unique insight. While many methods have been developed to study noncovalent interactions, symmetry-adapted perturbation theory (SAPT) stands out as one of the most robust. In addition to providing energetic information about an interaction, it provides insight into the underlying physics of the interaction by decomposing the energy into electrostatics, exchange, induction and dispersion. Therefore, SAPT is capable of not only answering questions about how strongly a complex is bound, but also why it is bound. This proves to be an invaluable tool for the understanding of noncovalent interactions in complex systems. The wavefunction-based formulation of SAPT can provide qualitative results for large systems as well as quantitative results for smaller systems. In order to extend the applicability of this method, approximations to the two-electron integrals must be introduced. At low-order, the introduction of density fitting approximations allows SAPT computations to be performed on systems with up to 220 atoms and 2850 basis functions. Higher-orders of SAPT, which boasts accuracy rivaling the best theoretical methods, can be applied to systems with over 40 atoms. Higher-order SAPT also benefits from approximations that attempt to truncate unneccesary unoccupied orbitals.

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The studies discussed in this thesis unify experimental and theoretical techniques, both established and novel, in investigating the problem of how a protein that binds specific sites on DNA translocates to, recognizes, and stably binds to its target site or sites. The thesis is organized into two parts. Part I outlines the history of the problem and the theory and experiments that have addressed the problem and presents an apparent incompatibility between efficient search and stable, specific binding. To address this problem, we elaborate a model of protein-DNA interaction in which the protein may bind DNA in either a search (S) mode or a recognition (R) mode. The former is characterized by zero or weak sequence-dependence in the binding energy, while the latter is highly sequence-dependent. The protein undergoes a random walk along the DNA in the S mode, and if it encounters its target site, must undergo a conformational transition into the R mode. The model resolves the apparent paradox, and accounts for the observed speed, specificity, and stability in protein-DNA interactions. The model shows internal agreement as regards theoretical and simulated results, as well as external agreement with experimental measurements. Part II reports on research that has tested the applicability of the two-mode model to the tumor suppressor transcription factor p53. It describes in greater depth the experimental techniques and findings up to the present work, and introduces the techniques and biological system used in our research. We employ single-molecule optical microscopy in two projects to study the diffusional kinetics of p53 on DNA. The first project measures the diffusion coefficient of p53 and determines that the protein satisfies a number of requirements for the validity of the two-mode model and for efficient target localization. The second project examines the sequence-dependence in p53's sliding kinetics, and explicitly models the energy landscape it experiences on DNA and relates features of the landscape to observed local variation in diffusion coefficient. The thesis closes with proposed extensions and complements to the projects, and a discussion of the implications of our work and its relation to recent developments in the field.

Group I introns are catalytic RNAs capable of self-splicing out of RNA transcripts. Ribozymes derived from these group I introns are used to explore the molecular recognition properties involved in intron catalysis. New ribozyme reactions are designed based on the inherent ability of these ribozymes to perform site-specific nucleophilic attacks. This study explores the molecular recognition properties of group I intron-derived ribozyme reactions and describe a new ribozyme reaction involving molecular recognition properties previously not seen.We report the development, analysis, and use of a new combinatorial approach to analyze the substrate sequence dependence of suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS) mediated reactions is not high, suggesting that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.A group I intron-derived ribozyme from P. carinii has been previously shown to bind an exogenous RNA substrate, splice-out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We now report that a group I intron derived ribozyme from the ciliate Tetrahymena thermophila can also perform the trans excision-splicing reaction, although not nearly as well as the P. carinii ribozyme.In addition, we discovered a new ribozyme reaction called trans insertion-splicing where the P. carinii ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Although this reaction gives the reverse products of the trans excision-splicing reaction, the trans insertion-splicing reaction is not simply the reverse reaction. The ribozyme recognizes two exogenous substrates through more complex molecular recognition interactions than what has been previously seen in group I intron-derived ribozyme reactions. We give evidence for this new reaction mechanism composed of three steps, with intermediates attached to the ribozyme.

Interactions between carbohydrates and proteins are increasingly being recognized as crucial in many biological processes, such as cellular adhesion and communication. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored. To date, several methods have been exploited to analyze interactions of carbohydrates and proteins, for example, biosensors, nuclear magnetic resonance (NMR); enzyme-linked immunosorbent assays (ELISA), X-ray crystallography and array technologies. This thesis describes the development of novel strategies for the synthesis of carbohydrates, as well as new efficient strategies to Quartz Crystal Microbalance- (QCM-) biosensors and carbohydrate microarrays technologies. These methodologies have been used to probe carbohydrate-lectin-interactions for a range of plant and animal lectins. / QC 20100915

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: David Andrews. Includes bibliographical references (leaves 206-242). Also available via World Wide Web.

Proefschrift Rijksuniversiteit Groningen.

xx, 281 p. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / More than two-thirds of all enzyme substrates and cofactors are anionic, emphasizing the essential role that anions play in biological processes. Moreover, anions can have detrimental effects on the environment by causing ground water contamination when anions such as perchlorate, phosphate and nitrate develop in intolerable levels. Owing to the prevalent nature of anions, traditional strategies employed to target anions--including hydrogen bonding, metal ion coordination and electrostatic interactions--have been extensively studied. An alternative approach to anion binding would complement the powerful array of existing techniques. Recently, in the supramolecular chemistry community, new insight has been cast on how anions attractively interact with electron-deficient arenes, suggesting that aromatic rings are a viable anion binding strategy to balance existing methods. Chapter I provides a historical perspective of anions interacting with electron- deficient arenes. This outlook has its origins in the late 1800s with the discovery of colored charge-transfer complexes between donor and acceptor molecules and continues with the progression of the field leading up to the recent supramolecular fascination. Chapter II represents our initial efforts at measuring anion/arene interactions in solution. In particular, sulfonamide based hydrogen bonding receptors were developed with pendant aromatic rings to test the strength of anion/arene interactions in solution. Complementary computational chemistry and crystallography were utilized to supplement the solution studies. Chapter III describes our quantum calculations and crystallographic efforts at using only electron-deficient arenes to bind halides. A Cambridge Structure Database survey supports our emphasis of understanding multiple anion/arene interactions. Chapter IV illustrates how tripodal anion receptors can be developed to bind anions using only electron-deficient aromatic rings. Furthermore, subtle changes in anion binding geometries are observed with isomeric receptors and corroborated with Density Functional Theory calculations. Chapter V is dedicated to the preparation of electron-deficient anion receptors that are conformationally stabilized by hydrogen bonds. Chapter VI is committed to using our knowledge of anion binding to study a series of ethynyl-pyridine sulfonamides capable of hydrogen bonding to small molecules and anions. In conclusion, Chapter VII is a summary and future prospective for the field of anion/arene interactions. This dissertation includes previously published and co-authored material. / Adviser: Darren W. Johnson

In this thesis, a polypeptide binder concept is illustrated. By conjugation to a set of sixteen polypeptides, a small binding molecule can evolve into a polypeptide binder with increased affinity and selectivity. The concept was applied to 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) and acetazolamide (AZM) for development of high affinity binders targeting human C-reactive protein (CRP) and human carbonic anhydrase (HCA) II and IX respectively. In addition, peptididic macrocycles were developed as inhibitors of lysine specific demethylase 1 (LSD1). CRP is a well-known biomarker of inflammation in humans and binders recognizing it are therefore of large interest as medical diagnostics. Until now, phosphocholine (PCh) and derivatives are the only known small molecule binders for CRP, but they have low μM affinity and bind CRP in a Ca2+ dependent manner. The small molecule DQ was designed as a CRP binder that is structurally unrelated to PCh. Its polypeptide conjugate, 4-C25l22-DQ, was demonstrated as a strong, Ca2+ independent binder for CRP, and had an affinity approximately three orders of magnitude higher than DQ itself. HCA IX is a protein that is interesting for diagnosis of cancer. AZM is a small molecule inhibitor of HCAs with a dissociation constant of 38 nM for HCA II and 3 nM for HCA IX. Interestingly, polypeptide conjugate 4-C10L17-AZM displayed stronger binding to both HCA II (KD 4 nM) and HCA IX (KD 90 pM). This result provided evidence that the binder concept can be applied also for small molecules which already have high affinity for their protein receptors. LSD1 is an enzyme that regulates the methylation of Lys 4 of histone 3 via a PPI-like interaction and which is of therapeutic interest in certain cancers. Based on the structures of two peptidic ligands bound to LSD1, we sequentially prepared truncated, mono-substituted and macroyclic peptides in order to develop reversible inhibitors of LSD1. Some stapled cyclic peptides bound to LSD1 with 10-fold higher affinity than the corresponding linear parent peptide. Changing the staple into a lactam further improved the binding potency and the best lactams inhibited the enzymatic activity of LSD1 at low μM Ki values.

Molecular recognition is essential to all biological interactions and processes. Knowledge of the structural basis of recognition offers a powerful mechanism for understanding, predicting and controlling the behaviour of biological systems. In this thesis, we present, firstly a computational and crystallographic analysis of molecular recognition in protein-ligand systems; and secondly, progress towards the synthesis of a fluorescent probe for calcium ion recognition. Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kγ, have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders. As a step towards improved understanding of PI3K binding preferences, we examine the basis on which PI3Kγ distinguishes γ-selective inhibitors AS-605240 and AS-604850, with a ~30-fold preference for the former. Interestingly, despite the chemical similarity of the two ligands, the X-ray structures for their PI3Kγ complexes exhibit the molecules in different conformers, s-cis for AS-604850 and s-trans for AS-605240. Here, we re-examine the PI3Kγ/AS-605240 crystallographic data and find that not only is a s-cis conformation possible but in fact it has a much higher occupancy (87%) than the originally modelled s-trans isomer (13%). Subsequently, to account for the isomeric complexities presented by the ligands, we perform 140 ns MD simulations of the four PI3Kγ complexes in explicit solvent: this reveals similar conformational flexibility at the active site for all systems. Yet, the conformations sampled by the s-cis isomers are more consistent with the conformations reported by the X-ray crystal structures. Subsequent energetic analysis was performed incorporating ensemble-averaging and desolvation effects via the Poisson-Boltzmann continuum solvent model. For both inhibitors the s-cis isomers are predicted to be the most favourable conformations. Additionally, the results indicate a preference for AS-605240, as observed experimentally. The molecular basis for this preference is discussed, together with a comparison of molecular mechanical and quantum chemical treatments of the key ligand-Val882 interaction. This study provides structural, dynamical and energetic insights into the subtle basis of molecular recognition by PI3Kγ.Fluorescent probes have evolved into an extremely useful tool for the detection of calcium in biological systems. Benzothiazole derivatives BTC, and its iminocoumarin analogue BTIC, are two low affinity calcium indicators featuring many desirable properties for cellular calcium measurement. In an effort to produce fluorophores that can be chemically conjugated with a screening protein, such as Green Fluorescent Protein (GFP), we aimed to derivatise BTC and BTIC. Two synthetic approaches towards the synthesis of these potential fluorescent probes are outlined.