A dissertation submitted in partial fulfilment of the requirements for the degree of Master of Technology: Chemistry, Durban Institute of Technology, 2004. / 4-amino-(D3)-trishomocubane-4-carboxylic acid (tris-amino acid) is a constrained a-amino acid residue that exhibits peculiar conformational characteristics. The aim of the present study is to provide a deeper understanding of these features, which can be used as a guide when chOOSing@shomocubane as suitable building blocks for peptide design. The Ca carbon of@ishomocubane forms part of the cyclic structure, and consequently a peptidic environment was simulated with an acetyl group on its N-terminus and a methyl amide group on its C-terminus. This study involved a complete exploration of the conformational profile of (Yishomocubane using computational techniques.The parm94 parametization of the AMBER oio forc@eld was used to explore the conformational space of the peptide,Q)\xEFshomocubane. The Ramachandran maps computed at the molecular mechanics level' with the parm94 forc@\xEFeld parameters compared reasonably with the corresponding maps computed at the Hartree Fock (HF) level, using the 6-31G* basis set. The results of this study revealed that the conformational profile of the @ishomocubane peptide can be characterized by four low energy regions, viz., C7ax, C7eq, 310 and al helical structures. / M

While protein α-N-terminal methylation has been known for nearly four decades since it was first uncovered on bacteria ribosomal proteins L33, the function of this modification is still not entirely understood. Recent discoveries have demonstrated α-N-terminal methylation is essential to stabilize the interactions between regulator of chromosome condensation 1 (RCC1) and chromatin during mitosis, to localize and enhance the interaction of centromere proteins (CENPs) with chromatin, and to facilitate the recruitment of DNA damage-binding protein 2 (DDB2) to DNA damage foci. Identification of N-terminal methyltransferase 1 (NTMT1) unveiled the eukaryotic methylation writer for protein α-N-termini. In addition, NTMT2 that shares over 50% sequence similarity, has been identified as another mammalian protein α-N-terminal methylation writer. Knockdown of NTMT1 results in mitotic defects and sensitizes chemotherapeutic agents in breast cancer cell lines, while NTMT1 knockout mice showed premature aging. Additionally, NTMT1 has been shown to be overexpressed in a colorectal and melanoma tumor tissues, and in lung and liver cancer cell lines. Given the vast array of clinical relevance, chemical probes and inhibitors for NTMT1 are vital to elucidate information about the function and downstream process of protein α-N-terminal methylation. Therefore, 47 peptidomimetic compounds have been synthesized that target NTMT1. These peptide-based compounds range from three to six amino acids in length and the top 5 compounds have 3- to 300- fold selectivity for NTMT1 compared to other methyltransferases. An inhibition mechanism study has also been performed to verify the inhibitors are targeting the NTMT1 peptide binding site. Seven compounds have an IC50 of less than 5 µM and our top inhibitor, BM-47, has an IC50 of 0.32 µM ± 0.06 for NTMT1. To further elucidate information about the NTMTs and their downstream effects, we utilized photoaffinity probes to target these enzymes. Our 6 photoaffinity probes exhibited in a dose- and time-dependent manner. Probe labeling has been shown to be driven by recognition and selectively and competitively label the NTMT writers in a complex cellular mixture. Our results also provided the first indication of substrate preferences among NTMT1/2. Methylated photoaffinity probes were also synthesized to identify novel proteins that recognize a methylated N-terminus and shed light on the function of α-N-terminal methylation.

Among multiple interconnected pathways for L-Lysine catabolism in pseudomonads, it has been reported that Pseudomonas aeruginosa PAO1 employs the decarboxylase and the transaminase pathways. However, knowledge of several genes involved in operation and regulation of these pathways was still missing. Transcriptome analyses coupled with promoter activity measurements and growth phenotype analyses led us to identify new members in L-Lys and D-Lys catabolism and regulation, including gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for putative L-Lys efflux and lysP for putative L-Lys uptake. The amaAB operon is induced by L-Lys, D-Lys and pipecolate supporting the convergence of Lys catabolic pathways to pipecolate. Growth on pipecolate was retarded in the gcdG and gcdH mutants, suggesting the importance of glutarate in pipecolate and 2-aminoadipate utilization. Furthermore, this study indicated links in control of interconnected networks of lysine and arginine catabolism in P. aeruginosa. Effect of D-amino acids and the genes involved in their metabolism are of great interest in both bacteria and mammals. D-Arg utilization in PAO1 requires the coupled dehydrogenases DauB and DauA. In this study, DauB was found to use only L-Arg as its substrate unlike its partner dehydrogenase DauA with wide substrate specificity. However, evidence from this study and previous studies suggest that the coupled enzymes DauB and DauA are unique for D-Arg catabolism. The three D-amino acid dehydrogenases DguA, DadA and DauA were found to have somewhat limited in vivo substrate specificity compared to that found in vitro tested using purified enzymes. Many studies showed that D-amino acids are toxic to bacteria. The ΔdguA, ΔdadA and ΔdauA triple mutant had two-fold lower minimum inhibition concentration of carbenicillin and tetracycline compared to wild-type PAO1. Both in the wild-type PAO1 and the triple mutant, synergy was observed between gentamicin or tetracycline (at concentrations below the MIC) and D-amino acids resulting in growth inhibition or reduction, respectively. However, no special synergistic or antagonistic effects were observed specifically in the ΔdguA, ΔdadA and ΔdauA triple mutant as compared to the wild-type PAO1 when D-amino acids were given in combination with antibiotics.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / The impact of computer simulations has become quite significant especially with the development of supercomputers during the last couple of decades. They are used in a wide range of purposes such as exploring experimentally inaccessible phenomena and providing an alternative when experiments are expensive, dangerous, time consuming, difficult and controversial. In terms of applications in biological systems molecular modeling techniques can be used in rational drug design, predicting structures of proteins and circumstances where the atomic level descriptions provided by them are valuable for the understanding of the systems of interest. Hence, the potential of computer simulations of biomolecular systems is undeniable. Irrespective of the promising uses of computer simulations, it cannot be guaranteed that the results will be realistic. The precision of a molecular simulation depends on the degree of sampling achieved during the simulation while the accuracy of the results depends on the satisfactory description of intramolecular and intermolecular interactions in the system, i.e. the force field. Recently, we have been developing a force field for molecular dynamics simulations of biological systems based on the Kirkwood Buff (KB) theory of solutions, not only with an emphasis on the accurate description of intermolecular interactions, but also by reproducing several physical properties such as partial molar volume, compressibility and composition dependent chemical potential derivatives to match with respective experimental values. In this approach simulation results in terms of KB integrals can be directly compared with experimental data through a KB analysis of the solution properties and therefore it provides a simple and clear method to test the capability of the KB derived force field. Initially, we have provided a rigorous framework for the analysis of experimental and simulation data concerning open and closed multicomponent systems using the KB theory of solutions. The results are illustrated using computer simulations for various concentrations of the solutes Gly, Gly₂ and Gly₃ in both open and closed systems, and in the absence or presence of NaCl as a cosolvent. Then, we have attempted to quantify the interactions between amino acids in aqueous solutions using the KB theory of solutions. The results are illustrated using computer simulations for various concentrations of the twenty zwitterionic amino acids at ambient temperature and pressure. Next, several amino acids were also studied at higher temperatures and pressures and the results are discussed in terms of the preferential (solute over solvent) interactions between the amino acids. Finally, we have described our most recent efforts towards a complete force field for peptides and proteins. The results are illustrated using molecular dynamics simulations of several tripeptides, selected peptides and selected globular proteins at ambient temperature and pressure followed by replica exchange molecular dynamics simulations of a few selected peptides.

Peptides are a promising class of therapeutic candidates due to their high specificity and affinity for cellular protein targets. However, peptides are susceptible to protease degradation and are typically not cell-permeable. In efforts to design more effective peptide drug discovery systems, investigators have discovered that incorporation of non-canonical amino acids (ncAAs) and macrocyclization overcome these limitations, making peptides more drug-like. In this work, we exploit the promiscuity of wild-type aminoacyl-tRNA synthetases (aaRSs) to ‘mischarge’ ncAAs onto tRNA and ribosomally incorporate them into peptides using a cell-free translation system. We have demonstrated the ability to incorporate five ncAAs into a single peptide with near-wild type yield and fidelity. We also demonstrated the in situ incorporation of ncAAs containing azide and alkyne functionalities, enabling the use of CuAAC (click chemistry) to generate triazole-bridged cyclic peptides. When combined with bisalkylation of peptides containing two cysteines via an α,α’-dibromo-m-xylene linker, we created bicyclic peptides which are structurally similar to the highly bioactive knotted peptide natural products. Biological display methods, such as mRNA display, are powerful peptide discovery tools based on their ability to generate libraries of >1014 unique peptides. We combined our ability to incorporate ncAAs with our bicyclization technique adapted for use with mRNA display to create knotted peptide library scaffolds. We performed side-by-side monocyclic and bicyclic in vitro selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance. We used a new library that enables the generation of a diverse collection of linear, monocyclic and bicyclic scaffolds in one pot, increasing the likelihood of target-ligand conformational alignment. We performed a second selection against streptavidin and revealed a nearly unanimous preference for linear peptides containing an HPQ motif, a known streptavidin-binding sequence. However, when we used these libraries for in vitro selection against a biological target, DNA repair protein XRCC4, we did not observe convergence. In summary, we have developed a novel technique for production of bicyclic peptide libraries. These highly-constrained protease-stable scaffolds can be used as platforms to identify high affinity, drug-like ligands using mRNA display.

Diploma thesis is focused on creation of educational materials supporting the education of biochemistry, specifically amino acids and proteins, at secondary level. At first the analysis of Czech chemistry textbooks concerning the two topics - amino acids and proteins was performed. The major problems found were related to the insufficient graphical representation of biomolecules, unsatisfactory motivational components and insufficient integration of the topic with biology or everyday life experience. The supporting educational materials, presented in this work, supplement the widely used chemistry textbooks. The materials includes a graphic oriented presentation, interactive animations demonstrating the process of denaturation and precipitating of proteins at macroscopic and molecular level, poster presenting the structural formulas of standard amino acids, 3D models of selected proteins, additional texts supporting the current topics concerning amino acids and proteins and the laboratory protocols for students. The presented support materials were evaluated at the secondary school conditions. They were tested in one class and the improvement of student's understanding of the topic was compared to the second class employing only the classical educational methods. The comparison of the results of...

Master of Science / Department of Animal Sciences and Industry / Micheal J. Brouk / The study objective was to evaluate the application of supplemental rumen-protected lysine (RP Lys) to maintain milk production when reducing the crude protein levels in a lactating dairy cow diet. Twelve lactating multiparous Holstein cows, averaging 129 DIM, 50.2 kg milk yield, 3.6% fat and 2.9% true protein were randomly assigned to one of four 3x3 Latin squares. Each 14-d period had 11 d for adaptation followed by 3 d of data collection. Cows were offered one of three experimental treatment rations formulated with CPM Dairy (v3.0); Positive control (PC) — formulated to meet all nutrient requirements; Test diet (Test) — negative control diet formulated to meet nutrient requirements, except deficient in metabolizable protein (MP) (approximately 200 g/d) and first limiting in metabolizable Lys (approximately 10 g/d); and Test+RPL — same basal diet as negative control + RP-Lys to provide 14.5 g/d of MP-Lys. For Test+RPL, 45g of RP-Lys (AminoShure-L®; Balchem Corp., New Hampton, NY, containing 23.4g Lys) was top-dressed on the TMR once daily. The PC diet resulted in lower dry matter intake (P = 0.03) as compared to either the Test or Test+RPL diet. PC, Test, and Test+RPL cows averaged 42.6, 42.9, 43.6 kg/d of milk and 27.3, 28.4, 28.8 kg/d of DMI, respectively. Crude protein intake for the PC, Test, and Test+RPL diets was 4.83, 4.67, and 4.74 kg/d respectively. MUN decreased (P < 0.01) for cows on Test and Test+RPL diets as compared to PC diet (12.5, 12.5 and 14.9 mg/dL, respectively). Milk yield, milk components, milk component yields, FCM, ECM, SCM and production efficiencies (milk, ECM, SCM and FCM) did not differ (P > 0.05) among treatments. A post-study CPM Dairy evaluation using final chemical composition analyses of the feedstuffs and average production data from the animals predicted that diets supported more than 47 kg of milk and Lys was not limiting. Cows on the study produced slightly less milk, however DMI was 5-8% more than predicted by initial formulations. Formulation accuracy of the MP and Lys deficient diet may have been improved if data had been available from an initial adjustment period measuring DMI, body weight, milk yield and milk composition. It is also possible that the bioavailability of the RP Lys was not as great as thought during the diet formulation process. However, given the fact that the post-trial CPM analysis did not indicate a deficiency of Lys, it is not very likely that this impacted the results of this trial.

Marine phytoplankton are known to produce UV-absorbing compounds (UVACs) for protection against UV radiation. To assess whether the same strategy applies to sea ice-associated algal communities, MAAs were measured in algae associated with surface melt ponds, sea ice, sea ice−water interface, and underlying seawater in a coastal bay of the Canadian Arctic Archipelago during the 2011 spring melt transition. Six UVACs were detected as the spring melt progressed, namely shinorine, palythine, and porphyra-334 and three unknowns (U1, U2 and U3). U1 was most likely palythene, another MAA. The molecular identities of the other two UVACs, U2 and U3, which have an absorption maximum of 363 and 300 nm, respectively, remain to be structurally elucidated. The results confirm that Arctic sea ice-associated algal communities are capable of producing photoprotectants and that spatial and temporal variations in MAA and other UVAC synthesis are affected by snow cover and UV radiation exposure. / May 2016

The knowledge on the conformations of amino acids is essential to understand the biochemical behaviors and physical properties of proteins. Comprehensive computational study is focused to understand the conformational landscape of three aromatic amino acids (AAAs): tryptophan, tyrosine, and phenylalanine. Three different density functionals (B3LYP, M06-2X and wB97X-D) were used with two basis sets of 6-31G(d) and 6-31+G(d,p) for geometry optimizations of the conformers of AAAs followed by the vibrational frequencies. The goal was to identify the right choice of density functional theory (DFT) level for conformational analysis of amino acids by comparing the computational data against the available experimental results. Calculated infrared (IR) frequency values indicated that wB97X-D/6-31+G(d,p) level is less favorable than other DFT levels in case of O-H and N-H stretching frequencies for the conformers of AAAs. The C=O stretching frequencies at different computational levels were in good agreement with the experimental results. Interactions of AAAs (tryptophan, tyrosine, and phenylalanine) and two cyclic amino acids (histidine and proline) individually with two finite-sized graphene sheets (C62H20 and C186H36) were explored using M06-2X/6-31G(d) level. Computational investigations of the binding of amino acids with graphene provide knowledge for designing of new graphene-based biological/biocompatible materials. Selected conformers for each amino acid with different orientations on the surface of graphene were examined. The purpose of computational study on graphene-amino acids interactions was to identify the preferred conformer of amino acid to bind on graphene as well as to find the influence of amino acid binding on the band gap of graphene. Different conformers of AAAs generally prefer parallel orientation through π-π interactions to bind with graphene. However, bent orientation is more preferred over parallel to bind on the surface of graphene in case of conformer having relative energy approximately equal to 5 kcal/mol for all three AAAs. Histidine generally exhibits higher binding affinity than proline to form complex with graphene. The binding energies in the aqueous medium were slightly lower than those obtained in the gas phase with some exceptions. The adsorption of amino acids did not affect the band gap of graphene.

A síntese em fase sólida é o método mais utilizado na obtenção de peptídeos em laboratório. Em geral, as diferentes etapas do processo são realizadas em temperatura ambiente. De fato, até o início desta década, temperaturas elevadas (>40ºC) eram empregadas apenas esporadicamente nas etapas de acoplamento. Em 1993, entretanto, Rabinovich e Rivier propuseram a realização de todas as etapas do processo a 75ºC. Em 1996, Saneei e colaboradores tentaram automatizar o processo a 40-50ºC, apesar de não ter sido feita nenhuma avaliação sistemática prévia. Em 1997, Varanda & Miranda descreveram o primeiro estudo sistemático feito com o objetivo de investigar aspectos, vantagens, desvantagens e limitações da SPFS em altas temperaturas. O presente trabalho teve como objetivo ampliar os conhecimentos sobre o método através da avaliação de três importantes aspectos considerados problemáticos na SPFS convencional: racemização, agregação e síntese de peptídeos longos. Para tanto, vários peptídeos foram escolhidos como modelos de estudo. Todos eles foram sintetizados por SPFS convencional (usando protocolos de rotina de nosso laboratório) e por SPFS em alta temperatura (empregando as condições experimentais descritas por Varanda & Miranda em 1997). Os materiais brutos foram comparados em: 1- suas recuperações a partir de suas peptidil-resinas correspondentes; 2- suas qualidades. RP-HPLC, determinação do conteúdo de aminoácidos e CE foram os métodos de análise empregados. Os produtos e subprodutos majoritários foram isolados para caracterização química por análise de aminoácidos de seus hidrolisados totais e por espectrometria de massas. Os resultados obtidos demonstraram que no aspecto racemização a SPFS em alta temperatura, nas condições experimentais empregadas, mostrou-se equivalente à SPFS convencional: a incidência de estereoisômeros contaminantes nos peptídeos brutos foi inferior a 1%. O estudo do aspecto agregação evidenciou que se por um lado as condições experimentais empregadas para a SPFS em alta temperatura levaram a uma suposta minimização, por outro os peptídeos brutos obtidos foram de qualidade muito inferior a daqueles provenientes da síntese convencional: interrupções do crescimento das cadeias em resíduos de glutamina levaram à formação significativa de peptídeos contaminantes delatados. Se a agregação está ou não relacionada ao favorecimento destas reações secundárias é questão ainda não respondida. A síntese da CCK-22 humana não sulfatada sugeriu que o método de SPFS em alta temperatura é adequado à sua obtenção com bom rendimento e em tempo reduzido. Entretanto, a síntese da CCK-33 correspondente, um protótipo de peptídeo longo de seqüência complexa, evidenciou que, para a obtenção deste tipo de composto, a mistura 25%DMSO/tolueno pode não ser o solvente ideal comum para as etapas de desproteção, neutralização e acoplamento. Nestas condições, a mistura DIC/HOBt pode também não ser a melhor opção. / Stepwise solid-phase synthesis (SPPS) is the method of choice to prepare peptides. Although deprotection, neutralization and acetylation have been generally carried out at room temperature, the couplings have not. Indeed, elevated temperatures have been used to enhance coupling efficiency in conventional SPPS. The idea of performing all the synthetic steps at high temperatures, shortening the peptide chain build-up process, has not been explored until the 90s. In 1993, Rabinovich & Rivier proposed some modifications to the conventional SPPS protocols and achieved some peptide syntheses by performing deprotection, neutralization, coupling and acetylation at 75 ºC. In 1996, Saneei and co-workers tried to automate the entire synthetic process at 40-50 ºC. In 1997, Varanda & Miranda reported the first systematic study on SPPS at elevated temperatures. These authors determined optimized synthesis conditions of unsulfated cholecystokinin-12 by investigating aspects of the methodology such as temperature, solvent systems, resins compatibility, type of resins, peptide-resins swelling properties and, also, coupling agents efficiencies. The main goal of the present work was to evaluate SPPS at elevated temperature in three different aspects: racemization, aggregation and applicability to the synthesis of long and complex. peptides. For that, we chose some model peptides and synthesized them manually using conventional SPPS (our laboratory\'s routine protocols) and SPPS at elevated temperature (Varanda & Miranda\'s protocols). The crude peptides were compared in their recoveries from the corresponding peptidyl-resins after final cleavage and full deprotection and in their qualities. RP-HPLC, CE and amino acid analyses were the techniques employed in the comparisons. The major products and by-products were isolated and characterized using amino acid analysis and mass spectrometry. The results can be summarized as follows: 1- no significant contamination resulting from racemization has been detected in the crude peptides. The amounts of stereoisomers present on the crude peptides were always lower than 1% independently of the SPPS method employed or the analytical technique used for comparison; 2- no final conclusion could be taken regarding to the aggregation phenomenon. Under the experimental conditions employed for SPPS at elevated temperature, some couplings were definitely favoured. Despite that, the crude materials obtained showed low purities. Isolation and characterization of the major contaminants led us to conclude that the occurrence of undesired chain termination have also been favoured. Whether aggregation is related to that or not is to be investigated. 3- the SPPS at elevated temperature protocol employed showed to be suitable for the synthesis of unsulfated human CCK-22. The same can not be said for the unsulfated human CCK-33, a large and complex peptide. Thus, we concluded that for the preparation of this type of compound 25%DMSO/toluene and/or its combination with DIC/HOBt may not be a good choice.