Tubulin and elastin-like polypeptides (ELPs) both form large protein structures which can be thermodynamically evaluated using isothermal titration calorimetry and differential scanning calorimetry. ELPs are thermos-responsive biopolymers that undergo phase separation and form coacervates when heated. This project assesses the liquid-liquid phase separation of an ELP sequence derived from tropoelastin with a SynB1 cell-penetrating peptide attached to the N-terminus in conjunction with the chemotherapeutic drug doxorubicin. Microtubules (MTs) are a dynamic cellular structure formed of tubulin alpha/beta-heterodimers and are responsible for several important cellular processes, making them a viable target for anti-cancer drugs. There has been extensive research done to identify new ligands that show selective binding to microtubules. Ruthenium (II) polypyridyl complexes (RPCs) have been found to promote the polymerization of tubulin into microtubules. ITC has been used to determine the binding affinity of [Ru(II)(Ph2phen)3]2+ (RPC2).

<p>Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.</p> / Dissertation

The bonding in a series of ligand-bridged metal dimer complexes has been characterized by He(I) and He(II) photoelectron spectroscopy and approximate molecular orbital calculations. Bridging ligands such as carbonyl, nitrosyl, methylene and pyrazolyl in the complexes [CpFe(NO)]₂, [Cp*Fe(NO)]₂, [CpRu(NO)]₂, [Cp*Co(CO)]₂, [CpFe(CO)₂]₂, [Cp*Fe(CO)₂]₂, [CpFe(CO)]₂-μCO-μCH₂, [Cp*Fe(CO)]₂-μCO-μCH₂, [CpFe(NO)]₂- μCh₂, [CpRu(NO)]₂-μCH₂, [CpCo(CO)]₂-μCH₂, [CpRh(CO)]₂-μCH₂, [Ir(pyrazolyl)(CO)₂]₂, [Ir(3-methylpyrazolyl)(CO)₂]₂ and [Ir(3,5-dimethylpyrazolyl)(CO)₂]₂ are investigated and their effects upon metal-metal interactions are surveyed. Due to the presence of two d⁷ or d⁸ late metal atoms per molecule, these complexes display many overlapping ionization bands in a narrow valence ionization region. Attention has been given to modelling the photoelectron single ionization with asymmetric and symmetric Gaussians. The overlapping ionizations are successfully represented in terms of the model bandshapes. Thermodynamic relationships between bond dissociation and photoelectron ionization energies are also investigated. With relationships of this type, trends in bond energies may be correlated with ionization energies. Ligand inductive and bonding effects as well as small changes in molecular geometry cause shifts in the metal-based ionizations, which aid chemical understanding and interpretation of the molecular orbital picture. By comparing a series of related metal dimers, the assignment of related ionizations in the photoelectron spectra becomes apparent. Changes in ligand π accepting ability and changes in metal and formal oxidation states are also probed. Addition information is provided by vibrational fine structure in Cp₂Os, [CpFe(NO)]₂, and [Cp*Co(CO)]₂ and spin-orbit splitting in Cp₂Os. The metal-ligand backbonding combinations are found to be the most stable interactions and are responsible for the stability of the metal dimers. Metal-metal interactions are found to be relatively unimportant. Ligands with stronger π accepting abilities allow for more stabilized supported metal dimer complexes.

The kinetics for the bridge-to-chelate isomerization of the dppe ligand in H4Ru4(CO)10(dppe) have been investigated by UV-vis and NMR spectroscopies over the temperature range of 308-328 K. The isomerization of the ligand-bridged cluster 1,2-H4Ru4(CO)10(dppe) was found to be reversible by 31P NMR spectroscopy, affording a Keq = 15.7 at 323 K in favor of the chelating dppe isomer. The forward (k1) and reverse (k-1) first-order rate constants for the reaction have been measured in different solvents and in the presence of ligand trapping agents (CO and PPh3). On the basis of the activation parameters and reaction rates that are unaffected by added CO and PPh3, a sequence involving the nondissociative migration of a phosphine moiety and two CO groups between basal ruthenium centers is proposed and discussed. The substitution of the MeCN ligands in the activated cluster 1,2-Os3(CO)10(MeCN)2 by the diphosphine ligands dppbz proceeds rapidly at room temperature to furnish a mixture of bridging and chelating Os3(CO)10(dppbz) isomers and the ortho-metalated product HOs3(CO)9[μ-(PPh2)C=C{PPh(C6H4)}C4H4]. Thermolysis of the bridging isomer 1,2-Os3(CO)10(dppbz) under mild conditions gives the chelating isomer 1,1-Os3(CO)10(dppbz), molecular structure of both the isomers have been determined by X-ray crystallography. The kinetics for the ligand isomerization has been investigated by UV-vis and 1H NMR spectroscopy in toluene solution over the temperature range of 318-343 K. On the basis of kinetic data conducted in the presence of added CO and the Eyring activation parameters, a non-dissociative phosphine migration across one of the Os-Os bonds is proposed. Ortho metalation of one of the phenyl groups associated with the dppbz ligand is triggered by near-UV photolysis of the chelating cluster 1,1-Os3(CO)10(dppbz). The triosmium cluster 1,2-Os3(CO)10(MeCN)2 reacts with the diphosphine ligand 3,4­bis(diphenylphosphino)-5-methoxy-2(5)H-furanone (bmf) at 25 ºC to give the bmf-bridged cluster 1,2-Os3(CO)10(bmf). Heating 1,2-Os3(CO)10(bmf) leads to an equilibrium with the chelating isomer 1,1-Os3(CO)10(bmf). The molecular structure of each isomer has been crystallographically determined, and the kinetics for the isomerization has been investigated by UV-vis and 1H NMR spectroscopy. The reversible nature of the diphosphine isomerization has been confirmed by NMR measurements, and the forward (k1) and reverse (k-1) first-order rate constants for the bridge-to-chelate isomerization have been determined. Thermolysis of the SEQ CHAPTER h r 11,1-Os3(CO)10(bmf) cluster (>110 ºC) leads to regiospecific activation of C-H and P-C bonds, producing the hydrido clusters HOs3(CO)9[µ-PPh2C=C{PPh(C6H4)} CH(OMe)OC(O)] and the benzyne clusters HOs3(CO)8(μ3-C6H4)[µ-PPhC=C(PPh2)CH(OMe)OC(O)]. The hydride and benzyne clusters, which exist as a pair of diastereomers, have been fully characterized in solution by IR and NMR spectroscopy, and the molecular structure of one benzyne cluster (major diastereomer) has been determined by X-ray crystallography.

The Notch signalling pathway is universally conserved in all metazoan species, and is involved in many aspects of cell fate determination and tissue homeostasis, during development and in adult organisms. Several developmental diseases are associated with defective Notch signalling, and the Notch pathway has been implicated in a growing number of cancers. The Notch signalling pathway requires direct cell-cell contact for ligand binding and receptor activation to occur. Specific domains within the Notch receptors and ligands have been identified as necessary for the interaction to take place, and a series of enzymes are known to regulate Notch signalling via glycosylation. Other domains beyond the minimal ligand binding region of the Notch receptor are also known to influence binding. The aim of this study was to characterise the molecular basis for ligand binding by the Notch receptor, and how this is regulated by glycosylation. The effects on ligand binding of specific amino acid substitutions and sugar modifications were tested using prokaryotically -expressed proteins, and a series of constructs containing additional domains N-terminal of the ligand binding region was produced prokaryotically and eukaryotically to test how additional domains might affect ligand binding. Binding was assessed by a flow cytometry-based binding assay and by SPR in order to investigate how particular modifications affected ligand binding. These assays indicated that an evolutionarily-conserved hydrophobic site exists within the central β-sheet of EGF12 in the Notch receptor that is directly adjacent to the O-fucosylation site within this domain. The GlcNAc-fucose disaccharide modification at this position was found to increase binding of hNotch1 to both Jagged1 and DLL4. Additional EGF domains N-terminal to the ligand binding region showed opposite effects on binding to these two ligand classes, suggesting that the precise mode of binding may vary slightly between different Notch ligands.

Phosphate transfer is ubiquitous in nature, however the occurance of phosphomutases is rare. Their uniqueness can be attributed to the complex and malleable substrate recognition scheme that allows the enzyme to perform two similar, yet distinct, catalytic steps while maintaining strict fidelity for substrate versus water. The complexity of developing this mechanism is highlighted in that, while phosphomutase function has independently evolved in most larger phosphotransferase superfamilies, very little diversification of this function has developed. As such, phosphomutases provide a rich framework to study the intricate specificity mechanisms employed by enzymes. β-Phosphoglucomutase (bPGM) catalyzes the interconversion between β-glucose 1-phosphate (βG1P) and glucose 6-phosphate (G6P) via a β-glucose 1,6 bisphosphate (βG16P) intermediate. βPGM is in one of two subfamilies that have independently acquired phosphomutase activity within the ubiquitous Haloalkanoate Dehalogenase superfamily (HADSF) of phosphotransferases. The enzyme has been observed to undergo a large conformational change upon binding βG16P as well as a repositioning of the general acid/base catalyst residue Asp10. In addition, the mechanism involves cycling of the protonation state of Asp10, which requires a significant pKa shift. The importance of Asp10 and its activation of the enzyme have been discussed previously, however a clear understanding of the interplay between the conformational and catalytic activation mechanisms for βPGM has not been described. This work uses aqueous phase techniques, solution X-ray scattering and molecular dynamics, to probe the effect of individual ligand moieties on the conformational state of the enzyme and free energy molecular dynamics and electrostatic calculations determine the interplay between conformation, protonation and Asp10 activation. The results implicate a model where the ligand-induced conformational change is governed by the non-catalytic phosphate site, and this transition induces correct positioning of Asp10, which, in turn induces the pKa shift, forming the catalytically competent complex.

In chapter 1, I discuss how ligand architecture in tripodal nitrogen-rich ligands can drastically affect the structure of zinc complexes featuring these ligands. The synthesis and characterization of zinc tris(1-methylimidazol-2-ylthio)methyl ([Titm^Me]) and tris(1-Pribenzimidazol-2-ylthio)methyl ([Titm^iPr,benzo]) complexes is presented. The ligand in [Titm^Me]Zn complexes binds the metal to form carbatrane structures that exhibit unusually long and flexible Zn–C bonds. The bonding between the zinc and the carbon in these complexes can therefore be more accurately described as a zwitterionic interaction between a carbanion and a zinc cation. Density functional theory calculations demonstrate that the energy profile for the Zn–C bond is shallow, such that large variations of the Zn–C distance result in very little change in the energy of the complex. The benzannulated ligand [Titm^iPr,benzo] allows access to a rare monomeric zinc hydride species [κ³-Titm^iPr,benzo]ZnH that can react with either CO₂ to produce a zinc formate, or B(C₆F₅)₃ to form the ion pair [κ⁴-Titm^iPr,benzo]ZnHB(C₆F₅)₃. The coordination chemistry of the [Titm^iPr,benzo] ligand also extends to the other metals of group 12. In chapter 2, I report the use of the [Titm^Me] and [Titm^iPr,benzo] zinc complexes presented in chapter 1 as biomimetic models for zinc enzymes. First, [Titm^Me] zinc complexes present structural similarities with the active site of carbonic anhydrase, and can be used to study the binding of carbonic anhydrase inhibitors to the enzyme active site. Then, [κ⁴-Titm^iPr,benzo]ZnX (X = MeB(C₆F₅)₃, BPh₄) complexes and their interactions with ligands of relevance towards antibiotic resistance is reported. The non coordinating nature of the anions in [κ⁴-Titm^iPr,benzo]ZnX (X = MeB(C₆F₅)₃, BPh₄) lead to the formation of a Lewis acidic zinc cationic center, which can be coordinated by an additional ligand of biological interest. The binding of simple β-lactams to the [κ⁴-Titm^iPr,benzo]ZnX complexes can be probed using X-ray diffraction and Nuclear Magnetic Resonance (NMR) spectroscopy, thereby providing a way to model the binding of antibiotics to the active site of the metallo-β-lactamases enzymes responsible for broad antibiotic resistance. The binding of β-lactams can be compared to larger ring size lactams and linear amides. [κ⁴-Titm^iPr,benzo]ZnX (X = MeB(C₆F₅)₃, BPh₄) also allows for the study of the binding of potential metallo-β-lactamases inhibitors, such as, for example, glycinamide, picolinamide, and piperazine-2,3-dione. Binding studies between [κ⁴-Titm^iPr,benzo]ZnX and substrates bearing structural similarities to antibiotics reveal secondary interactions involving peripheral functional groups the cationic zinc center in [κ⁴-Titm^iPr,benzo]ZnX. These studies provide guidelines to modify existing antibiotics, in order to decrease their sensitivity to metallo-β-lactamases. In chapter 3, I explore the reactivity of previously characterized tris(2-pyridylthio)methyl [Tptm] zinc complexes. First, an improved synthesis of [κ⁴-Tptm]ZnF using Me₃SnF as the fluorinating agent is reported. The fluorine atom in [κ⁴-Tptm]ZnF acts as a Lewis base, as illustrated by its reaction with B(C₆F₅)₃ to form [κ⁴-Tptm]ZnFB(C₆F₅)₃, in which the fluorine is transferred to the borane group. The fluoride ligand in [κ⁴-Tptm]ZnF also acts as a hydrogen bond and halogen bond acceptor and is capable of forming adducts with H₂O, indole, and iodopentafluorobenzene. [κ⁴-Tptm]ZnF undergoes metathesis with Ph₃CCl to form Ph₃CF, thereby providing a rare example of C–F bond formation promoted by a zinc complex. Then, [κ³-Tptm]ZnH is used as a catalyst for the hydrosilylation of aldehydes and ketones using phenylsilane to produce tris alkoxysilane products. The catalyst is very active with aldehydes, and shows slower reactivity towards dialkyl ketones. The reaction proceeds via insertion of the carbonyl group in the Zn–H bond to form a zinc alkoxide, which then undergoes metathesis with the silane to generate the desired product and regenerate the zinc hydride species. The complicated NMR spectroscopic features of the products resulting from the hydrosilylation of prochiral ketones are explained by the presence of different diastereomers. Finally, we report that [κ³-Tptm]ZnH is a catalyst for the hydrosilylation of silylformates to methoxy silanes with (EtO)₃SiH, (MeO)₃SiH and κ⁴-N(CH₂CH₂O)₃SiOMe. We show that CO₂ can be reduced to methoxy silane species in a one pot reaction using (MeO)₃SiH and catalytic amounts of [κ³-Tptm]ZnH. In chapter 4, I report the synthesis and characterization of a silicon based analogue of [Titm^iPr,benzo], namely the tris(1-Pribenzimidazol-2-yldimethylsilyl)methyl [Tism^iPr,benzo] ligand. The ligand possesses unique structural features, due to the proximity between the dimethylsilyl groups and the methyl carbanion. The formation of [κ⁴-Tism^iPr,benzo]Li proceeds via the doubly base stabilized silene intermediate [κ³-C(SiMe₂benzimid^iPr)₂]SiMe₂. [κ⁴-Tism^iPr,benzo]Li can be used as a precursor for copper and nickel [Tism^iPr,benzo] and [C₃-Tism^iPr,benzo] complexes, where [C3-Tism^iPr,benzo] represents the isomerized tris carbene version of [Tism^iPr,benzo]. [κ³-C(SiMe₂benzimid^iPr)₂]SiMe₂ reacts with ZnMe₂ to produce [κ³-C(SiMe₃)(SiMe₂benzimid^iPr)₂]ZnMe, which can be transformed to the phenoxide compound. This compound acts as a catalyst for the hydrosilylation of CO₂ to silyl formates and methoxy silanes. [κ³-C(SiMe₂benzimid^iPr)₂]SiMe₂ itself reacts with CO₂ to produce an unusual β-lactone.

Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar. / doctoral

Haemonchus contortus is a parasitic nematode that is controlled in large part by nematocidal drugs that target receptors of the parasitic nervous system. Hco-UNC-49 is a nematode GABA receptor that has a relatively low overall sequence homology to mammalian GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. However, the nematode receptors do exhibit different sensitivities to GABA which may be linked to differences in the putative GABA binding domains. Mutational analysis conducted in this study identified at least one amino acid, positioned near the GABA binding domain, which may partially account for differences in nematode GABA sensitivity. In addition, positions reported to be crucial for GABA sensitivity in mammalian receptors also affect GABA sensitivity in Hco- UNC-49 suggesting that the GABA binding domains of the mammalian and nematode GABA receptors share some pharmacological similarities. However, there were some differences observed. For example, in mammalian GABAA receptors amino acids from both  and  subunits appear to be important for GABA sensitivity. For residues examined in this study, only those on the UNC-49B subunit, and not UNC-49C, appear important for GABA sensitivity. / UOIT

No description available.