Дисертації з теми "Β-strand"

The work in this thesis reports studies directed to developing a calpain cysteine protease inhibitor that could be of value in slowing cataract development in humans. The work focuses on the development of macrocyclic compounds which can have advantages over acyclic compounds due to their resistance to proteolytic hydrolysis, improved selectivity, bioavailability and membrane permeability. A review of X-ray crystal structures of natural and synthetic calpain inhibitors complexed with the cysteine protease calpain show the inhibitors generally bind in the enzyme active site in an extended β-strand conformation. The calpain inhibitor SJA-6017 has been identified as a suitable lead compound. The importance of the para-fluoro group in SJA-6017 has been investigated. Modifications have been made to constrain this basic structure within a macrocycle and restrict the peptide chain as a β-strand conformation. Macrocycle CAT811 is a potent calpain 1 and 2 inhibitor and shows promise in slowing the progression of cortical cataract in trials with sheep having a hereditary propensity towards the development of cataract. In this thesis I report studies directed to improve the yield of the key RCM macrocyclisation step in the synthesis of aldehyde CAT811 and of three ester analogues (2.1, 2.3 and 2.4). I also report the development of a more commercial route to CAT811 not involving RCM but using intramolecular nucleophilic cyclisation. This intramolecular nucleophilic cyclisation strategy was attempted for the preparation of a histidine containing macrocyclic ester (4.1a) but was unsuccessful. An alternate strategy involving intramolecular lactamization proved successful for the synthesis of histidine-based macrocyclic esters (4.1a-4.3a). Reduction to the corresponding alcohols (4.1b-4.3b) was successful and oxidation of (4.1b and 4.3b) afforded the corresponding aldehydes (4.1c and 4.3c) for biological assay against ovine calpain 2. Aldehyde 4.3c has an IC50 of 1 μM and the corresponding alcohol 4.3b shows no activity (IC50 > 50 μM) consistent with the modelling which indicated that these two compounds did not adopt a β-strand conformation in the docking studies. Aldehyde 4.1c, on the other hand, shows significant inhibitory activity with an IC50 of 238 nM but as expected the corresponding alcohol 4.1b shows little activity (IC50 = 29 μM). Modelling studies showed that both the aldehyde 4.1c and the alcohol 4.1b on docking can form a β-strand with appropriate H-bonding interactions. The aldehyde is more active than the alcohol due to the reactivity of the aldehyde warhead allowing for the reversible formation of a hemiacetal. A similar difference in reactivity is observed for CAT811 (30 nM) and its alcohol analogue (700 nM). These results demonstrate the value of molecular modelling as a screening mechanism before unproductive synthetic work is considered.

Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC₅₀ of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC₅₀ against calpain II of 27 nM. The best non-electrophilic warhead macrocycle (5.13) had an IC₅₀ against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.

DNA polymerase epsilon (Pol ε) is a multi-subunit B-family DNA polymerase that is involved in leading strand DNA replication in eukaryotes. DNA Pol ε in yeast consists of four subunits, Pol2, Dpb2, Dpb3, and Dpb4. Pol2 is the catalytic subunit and Dpb2, Dpb3, and Dpb4 are the accessory subunits. Pol2 can be further divided into an N-terminal catalytic core (Pol2core) containing both the polymerase and exonuclease active sites and a C-terminus domain. We determined the X-ray crystal structure of Pol2core at 2.2 Å bound to DNA and with an incoming dATP. Pol ε has typical fingers, palm, thumb, exonuclease, and N-terminal domains in common with all other B-family DNA polymerases. However, we also identified a seemingly novel domain we named the P-domain that only appears to be present in Pol ε. This domain partially encircles the nascent duplex DNA as it leaves the active site and contributes to the high intrinsic processivity of Pol ε. To ask if the crystal structure of Pol2core can serve as a model for catalysis by Pol ε, we investigated how the C-terminus of Pol2 and the accessory subunits of Pol ε influence the enzymatic mechanism by which Pol ε builds new DNA efficiently and with high fidelity. Pre-steady state kinetics revealed that the exonuclease and polymerization rates were comparable between Pol2core and Pol ε. However, a global fit of the data over five nucleotide-incorporation events revealed that Pol ε is slightly more processive than Pol2 core. The largest differences were observed when measuring the time for loading the polymerase onto a 3' primer-terminus and the subsequent incorporation of one nucleotide. We found that Pol ε needed less than a second to incorporate the first nucleotide, but it took several seconds for Pol2core to incorporate similar amounts of the first nucleotide. B-family polymerases have evolved an extended β-hairpin loop that is important for switching the primer terminus between the polymerase and exonuclease active sites. The high-resolution structure of Pol2core revealed that Pol ε does not possess an extended β-hairpin loop. Here, we show that Pol ε can processively transfer a mismatched 3' primer-terminus between the polymerase and exonuclease active sites despite the absence of a β-hairpin loop. Additionally we have characterized a series of amino acid substitutions in Pol ε that lead to altered partitioning of the 3'primer-terminus between the two active sites. In a final set of experiments, we investigated the ability of Pol ε to displace the downstream double-stranded DNA while carrying out DNA synthesis. Pol ε displaced only one base pair when encountering double-stranded DNA after filling a gap or a nick. However, exonuclease deficient Pol ε carries out robust strand displacement synthesis and can reach the end of the templates tested here. Similarly, an abasic site or a ribonucleotide on the 5'-end of the downstream primer was efficiently displaced but still only by one nucleotide. However, a flap on the 5'-end of the blocking primer resembling a D-loop inhibited Pol ε before it could reach the double-stranded junction. Our results are in agreement with the possible involvement of Pol ε in short-patch base excision repair and ribonucleotide excision repair but not in D-loop extension or long-patch base excision repair.

Despite the unfavorable pharmacokinetic properties associated with peptides, they are still of great interest in drug development due to a multitude of interesting biological functions. The development of peptidomimetics strives to maintain or improve the biological activity of a peptide concurrently with removing the unwanted properties. This thesis describes two synthetic approaches to peptidomimetics with particular emphasis on secondary structure mimetics. First the design, synthesis and evaluation of two beta-turn mimetics incorporated in the endorphin Leu-enkephalin is presented. The beta-turn mimetics were stabilized by replacement of the intramolecular hydrogen bond with an ethylene bridge, and the amide bond between Tyr and Gly was replaced with an ether linkage. Linear analogues of the two mimetics were also synthesized. The peptidomimetics and their linear analogues were evaluated in a competitive binding assay at two opiate receptors, my and delta. One of the cyclized beta-turn mimetics was found to be a delta receptor antagonist with an IC50 value of 160 nM. Second a synthetic strategy to a beta-strand mimetic using 2-fluoro-4-iodopyridine as scaffold is described. The synthesis involved a Grignard exchange reaction on the pyridine scaffold using an amino acid derivative as electrophile followed by an SNAr reaction using an amine as nucleophile. The synthesis of a tripeptidomimetic of Leu-Gly-Gly and attempts to introduce chiral building blocks at the C-terminal, as well as studies towards elongated mimetics are presented. Two additional studies deal with the synthesis of two classes of potential thrombin inhibitors based on the pyridine scaffold. The first class contain pyridine as central fragment (P2 residue) substituted with a para-amidinobenzylamine group as P1 residue and various benzoyl groups as P3 residues. Three potential thrombin inhibitors were synthesized and found to be microM inhibitors in an enzymatic assay. In the second class, the pyridine ring serves as P3 residue. This class also lacks a strongly basic group in the P1 position. A small library of eight compounds were synthesized and evaluated in the enzymatic assay. Unfortunately, these compounds lacked inhibitory activity.

La kinase PKR (double stranded RNA dépendent protein kinase) est une kinase pro-apoptotique qui contrôle la traduction des protéines. Des études antérieures ont montré que la kinase PKR était activée dans les neurones en dégénérescence au cours de la maladie d'Alzheimer (MA). La kinase GSK3 bêta participe à la phosphophorylation de la protéine tau qui compose les « neurofibrillary tangles » au cours de la MA. PKR peut activer indirectement GSK 3 bêta dans les cultures de cellules. Nous montrons dans ce travail que PKR, est colocalisée avec GSK 3 bêta et tau phosphorylée dans les neurones des cerveaux de patients MA. Dans les cultures ed celules neurales SH-SY5Y , le stress du réticulum endoplasmique induit par la tunicamycin ou la neurotoxicité du peptide A bêta 1-42, active PKR, induit ractivation de GSK3, produit la phosphorylation de tau et conduit à l'apoptose. Les inhibiteurs pharmacologiques de PKR ou les si RNA de PKR atténuent les effets induits par ces stress dans les cultures de cellules. PKR pourrait représenter une nouvelle cible thérapeutique capable de diminuer la mise en jeu de processus pathologiques au cours de la MA conduisant à la dégénérescence neuronale
PKR or double-stranded RNA dependent kinase is a pro-apoptotic kinase that controls protein translation. Previous studies revealed that activated PKR is increased in AD brains. Glycogen Synthase Kinase Aβ(CS K3β) is responsible for tau phosphorylation and PKR can indirectly activate GSK3β in cell cultures. The goal of this work was to détermine if PKR could simultaneously trigger GSK3J5 activation and tau phosphorylation and apoptosis. In AD brains, both activated kinases co-localized with phosphorylated tau in neurons. In SH-SY5Y cell cultures, tunicamycin and Api_42 activate PKR, GSK3β and induce tau phosphorylation which are both attenuated by PKR inhibitors or PKR siRNA. Our results demonstrate that PKR is able to modulate GSK3β activation, tau phosphorylation and apoptosis in neuroblastoma cells exposed to tunicamycin or Aβ PKR could represent a potent pharmacological target to attenuate neurodegeneration and tau phosphorylation

Chapter One introduces the concept of peptide 'secondary structure' with an emphasis on β-strand geometry in macrocycles. This structural design is crucial for targeting different proteases. The significance of the macrocylic β-strand ‘bioactive’ conformation is discussed in detail. In particular the exploitation of the conformationally constrained peptidomimetic macrocylic backbone, which is constrained by a number of synthetic approaches to lock the ‘bioactive’ conformation in place. Chapter Two describes simple and scalable methodology for the preparation of N-Cbz protected amino acids by reaction with Cbz-Cl which uses a mixture of aqueous sodium carbonate and sodium bicarbonate to maintain the appropriate pH. This method proceeds without the formation of by-products. The method is extended to large scale preparation of an intermediate zofenopril, an ACE inhibitor. Chapter Three describes new peptidic templates constrained into a β-strand geometry by linking acetylene and azide containing P₁ and P₃ residues of a tripeptide by Huisgen cycloaddition. The conformations of the macrocycles are defined by NMR studies and those that best define a β-strand are shown to be potent inhibitors of the protease calpain. The β-strand templates presented and defined here are prepared under optimized conditions and should be suitable for targeting a range of proteases and other applications requiring such geometry. Chapter four describes a new approach to non-covalent peptide-based nanotubular or rodlike structures, whereby the monomeric units are preorganised into a β-strand geometry that templates the formation of an extended and unusual parallel β-sheet rod-like structure. The conformational constraint is introduced by Huisgen cycloaddition to give a triazolebased macrocycle, with the resulting self-assembled structures stabilized by a well-defined series of intermolecular hydrogen bonds. Chapter Five the 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new CT-L specific proteasome inhibitors demonstrated high potency and specificity for cancer cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry was associated with decreased activity in vitro and reduced anticancer activity, suggesting that the proteasome prefers to bind a conformationally flexible ligand. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition. Chapter Six peptide derived protease inhibitors represent an important class of compounds with the potential to treat a wide range of serious medical conditions. Herein we describe the synthesis of a series of triazole containing macrocylic protease inhibitors preorganised in a β-strand conformation and evaluate their selectivity and potency against a panel of protease inhibitors. A series of acyclic azido-alkyne-based aldehydes is also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards Calpain II, Cathepsin L and S and the 26S proteasome chymotrypsin-like activity. Importantly, the first examples of potent and selective inhibitors of Cathepsin S were identified and shown to adopt a well-defined β-strand geometry by NMR, X-ray and molecular docking studies. Chapter Seven describes simple and efficient methodology for the selective acylation and alkylation of biotin at its 3′-nitrogen. This methodology is used to prepare of other biotin derivatives.
Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2012

This thesis describes the design, preparation, and testing of a range of protease inhibitors. Chapter One introduces the concept of peptidomimetics, and discusses how proteases almost universally bind their ligands in a β-strand conformation. The idea of constraining a compound into a biologically active conformation by the introduction of a ring or bridge is discussed. The technique of ring closing metathesis as a strategy for macrocyclisation is introduced. The chapter also discusses calpain and HIV proteases and their structures and implications in human disease. Chapter Two surveys the acyclic calpain inhibitors reported in the literature. A series of N-heterocyclic peptidic calpain inhibitors were docked in silico into an ovine m-calpain homology model using Glide, which revealed that compounds 2.60 – 2.67 all adopted a β-strand conformation upon binding. The modelling revealed low energy conformations of 2.60, 2.61 and 2.66 not in a β-strand geometry. The synthesis and testing of these inhibitors is described, with 2.63 displaying an IC₅₀ of 40 nM against m-calpain in an in vitro assay. Chapter Three describes the design and synthesis of the β-strand mimic macrocycle 3.8, which was prepared using ring closing metathesis. The chapter also describes the design of a number of calpain and HIV protease inhibitors that incorporate 3.8. Each inhibitor is designed to bind and inhibit a specific protease target. Chapter Four describes the synthesis and testing of a series of macrocyclic calpain and proteasome 20S inhibitors. The preparation of the aldehydes 3.9 and 3.10 by elaboration of the macrocycle 3.8 is described. As well, the preparation of 3.10 from the N-capped 4-fluorosulphonyl diene 4.4 is described. The most potent macrocycle in the series was 3.10, which displays an IC₅₀ against m-calpain of 2000 nM, and an IC₅₀ against the chymotrypsin like activity of proteasome 20S of 2 nM. Chapter Five describes the synthesis of a series of building blocks, and their use in the attempted preparation of the potential HIV protease inhibitor 3.12a, as well as the successful preparation of the potential HIV protease inhibitors 3.11 and 3.12b. Preliminary studies testing the biological activity of compounds 3.11, 3.12b and 5.21 found that they displayed a percentage inhibition of HIV-1 subtype B protease of 86, 63, and 26%, respectively. The Ki of 3.11 against HIV-1 subtype B protease was also determined to be 62 nM. The activity of 3.11 against HIV-1 protease establishes that the common macrocyclic core 3.8 can be incorporated into inhibitors of both calpain, and HIV-1 protease. Chapter Six describes the preparation of a key macrocycle by cross-metathesis. The preparation of 6.4 by cross-metathesis of the olefins 6.5 and 6.24 is described, as well as the elaboration of 6.4 to give the macrocycle 6.1. A systematic study of the cross-metathesis of the olefins 6.5, 6.6, 6.23 and 6.24 is described. Their percentage conversion to 6.4 was calculated using high performance liquid chromatography analysis. The highest conversion to 6.4 was found to be 60%, from the cross metathesis of an equimolar mixture of 6.6 and 6.23. Chapter Seven describes a multi-gram synthesis of the potent macrocyclic calpain inhibitor CAT0811. The key step in the synthesis is the base induced macrocyclisation of the iodopeptide 7.10 to give 7.6. The macrocycle 7.6 was also prepared by macrolactamisation of the pseudopeptide 7.9. The synthesis was found to be scalable, affordable and efficient, and removes the need for Grubbs’ 2nd generation catalyst (II).
Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2011

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A series of ruthenium and iridium complexes have been synthesized and characterized with 20 novel crystal structures discussed. The library of β-ketoiminato complexes has been shown to be active against MCF-7 (human breast carcinoma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma), and A2780cis (cisplatin-resistant human ovarian carcinoma) cell lines, with selected complexes’ being more than three times as active as cisplatin against the A2780cis cell line. Selected complexes were also tested against the noncancerous ARPE-19 (retinal pigment epithelial cells) cell line, in order to evaluate the complexes selectivity for cancer cells. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is overexpressed in cancer cells, and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anticancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant levels of cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double-strand DNA breaks or DNA cross-linking but induced significant levels of single-strand DNA breaks, indicating a mechanism of action different from that of cisplatin.
Lord RM, Hebden AJ, Pask CM, Henderson IR, Allison SJ, Shepherd SL, Phillips RM, McGowan PC

Glioblastoma multiforme (GBM) is a highly lethal and incurable cancer of the central nervous system and current therapies are challenged by GBMs invasive growth and chemo-radioresistance. Ca2+-permeable N-Methyl-D-aspartate receptors (NMDARs) are important for synaptic transmission of excitatory neurons and essentially regulate the plasticity of our brain via activation of several NMDAR-dependent signaling pathways. However, NMDARs have been shown to contribute to GBMs malignancy by promoting growth, survival and migration. Although the impact of NMDARs on GBM has been clearly demonstrated, the particular signaling pathways used by GBM are poorly known. The identification of the NMDAR signaling pathways used by GBM cells might therefore help to develop NMDAR-targeted cancer therapies which highly impact GBM cells but do not disrupt synaptic transmission in neurons. The NMDAR-dependent expression of early-response genes (ERGs) upon neuronal activity is essential for synaptic plasticity and the formation of long term memory. The expression of ERGs depends on NMDAR-induced DNA-double strand breaks (DSBs) in the transcriptional start site of these genes. Some neuronal ERGs encode for proto-oncogenes like cFos, suggesting that GBM cells might hijack NMDAR signaling pathways to promote proto-oncogenes expression. In order to investigate the impact of NMDAR-dependent ERG expression in GBM cells we intended to identify the hallmark of this NMDAR signaling pathway: The induction of NMDAR-dependent and Topoisomerase II β (Top2β) mediated DSBs in GBM cells. For this task we validated the expression of NMDARs and functional Ca2+ signaling in the LN229 GBM cell line, which revealed functional NMDAR signaling in LN229 cells. Immunofluorescence staining of the DSB marker 53BP1 showed that NMDARs activation induces DSBs in a subpopulation of GBM cells and that DSB induction depends on Top2β activity, which demonstrates an analogues NMDAR signaling pathway in GBM cells and neurons. Analysis of ERG expression revealed that NMDARs, the cAMP-responsive element binding transcription factor (CREB) and Top2β all contribute to the expression of cFos and the brain-derived neurotrophic factor (BDNF) in GBM cells. Inhibition of Top2β or NMDARs also impaired the expression of cFos in a primary GBM cell line. In a clonogenic survival assay knock-down of Top2β with siRNAs and inhibition of NMDARs decreased LN229 cells resistance to X-rays. Additionally, a newly discovered interplay of NMDAR signaling and IR damage response on the expression of BDNF and cFos might explain the high impact of NMDAR inhibition on radiosensitivity. Interestingly, inhibition of DNA-dependent protein kinase indicates that NMDAR-mediated transcription involves factors required in DSB repair, suggesting an important role for DNA repair in NMDAR-mediated transcriptional regulation. The results presented in this work demonstrate a functional Top2β-dependent NMDAR signaling pathway in GBM cells. The radiosensitizing effect of Top2β and NMDAR inhibition reveals that targeting NMDAR-dependent and Top2β-mediated ERG expression might be a promising strategy for GBM therapy.

The work presented in this thesis focusses primarily on the determination of protein structure at atomic resolution, with NMR spectroscopy as the principle investigative tool. The thesis is divided into four parts. Part I consists of Chapter 1 which provides an introduction to protein structure, folding and NMR spectroscopy. Part II, consisting of Chapters 2 and 3, describes the effects of aromatic interactions on nucleating structure in disordered regions of proteins, using variants of apo-cytochrome b5 as a model system. Part III consists of Chapter 4, which describes structural effects of introducing cross-strand disulfide bonds using variants of Thioredoxin. Part IV of this thesis consists of the Appendices A, B and C. Appendix A describes the purification and characterization of ilvM, the regulatory subunit of the E.coli enzyme AHAS II. Appendices B and C contain chemical shift information corresponding to Chapter 3 and Chapter 4 respectively. Part I : Introduction to protein structure, folding and solution structure studies Chapter 1 first gives a brief overview of protein structure followed by an introduction to protein folding, focussing on the forces involved in determining the final three-dimensional shape of the protein as well as the experimental and computational techniques involved in studying or predicting the fold of a given protein. The second section of this chapter details the methodology followed to obtain solution structures of proteins using NMR spectroscopy. Part II : Engineering aromatic interactions to nucleate folding in intrinsically disordered regions of proteins Chapter 2 describes site-specific mutagenesis, recombinant over-expression, purifica-tion and preliminary biophysical characterization of two aromatic mutants of the molten globule apo-cytochrome b5 (apocytb5) : H43F H67F cytochrome b5 (FFcytb5) and H43W H67F cytochrome b5 (WFcytb5). Analysis of the structure of wild-type apo - cytochrome b5 was done to introduce surface mutations and avoid perturbation of the interior pack-ing of the protein. The bacterial host E.coli BL21(DE3) was used for recombinant over-expression, and both mutant proteins were purified by anion-exchange chromatography followed by size-exclusion chromatography. Biophysical studies show a decrease in the hydrodynamic radii and surface hydropho-bicity of FFcytb5 and WFcytb5 compared to wt -apo cytb5. An increase in protein stability was also seen from the wt apocytb5 to WFcytb5 and FFcytb5 in the presence of the chemical denaturant Urea. Proton 1D NMR spectra exhibited sharp lines and good spectral dispersion in the amide region, indicating that both mutant proteins are well folded. In addition, conservation of two distinctive up field and downfield shifted resonances present in apocytb5 indicated that structural changes upon mutation accrued on the upon the scaffold of apocytb5. Chapter 3 describes solution structure studies to determine secondary and tertiary structure of FFcytb5 and WFcytb5. Structural studies were carried out using homonu-clear and heteronuclear NMR methods, for which isotopically enriched 15N- and 13C, 15N samples were prepared for each protein. Additionally a 2H, 13C, 15N ILV methyl labeled sample was prepared for FFcytb5 to obtain unambiguous NOE correlation data. The hydrogen bond network for WFcytb5 was determined using hydrogen/deuterium exchange data. The restraints required to define the orientations and interactions of the aromatic groups were obtained from 15N-edited NOESY HSQC, 13C -edited NOESY HSQC and 2D 1H - 1H NOE spectra. These correlations were crucial in determining the aromatic interactions present within each protein. The structure of FFcytb5 was calculated using 1163 NOE distance restraints, 179 φ and ψ dihedral angle restraints, along with 40 hydrogen bond restraints. Similarly the structure of WFcytb5 was calculated using 1282 NOE distance restraints, 177 φ and ψ dihedral angle restraints and 40 hydrogen bond restraints. The ensemble of structures obtained for FFcytb5 showed a root mean square deviation of 1.01±0.21 Å . The ensemble of structures obtained for WFcytb5 showed a root mean square deviation of 0.58±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were found to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The final tertiary structure of both FFcytb5 and WFcytb5 consisted of a mixed four strand β -sheet with a four helix bundle resting on top and were seen to align well, with an RMSD of 0.6 Å. A comparison of the solution structures of apocytb5 with FFcytb5 and WFcytb5 convincingly showed the nucleation secondary and tertiary structure well beyond the site of mutation. The presence of aromatic trimers, non-canonical in context of the wt apoc-ytb5, was confirmed upon analysis of the structures of FFcytb5 and WFcytb5, with NOE correlations assigned to verify these interactions. The reduction in the hydrodynamic radii of FFcytb5 and WFcytb5 in relation to apocytb5 was also verified from tsuperscript15N-NMR relaxometry studies. The nucleation of long-range structure using aromatic interactions has been demonstrated in proteins for the first time, and can in principle be used to incorporate aromatic residues and interactions in protein design. Structural data, chemical shift data and restraints lists used for structure calculation of WFcytb5 and FFcytb5 were deposited with the PDB (accession numbers 5XE4 and 5XEE) and BMRB(accession numbers 36070, 36071) respectively1. Part III : Structural consequences of introducing disulfide bonds into β - sheets Chapter 3 describes the solution structure studies on two mutants of E.coli Thiore-doxin which were designed to incorporate a disulfide bond between two anti-parallel β-strands at the edge of the β-sheet. One mutant was designed with a disulfide bond at the hydrogen bonding position (HB, 78c90cTrx) and the other with the disulfide bond at the non-hydrogen bonding position (NHB, 77c91cTrx). Here we study the structural changes that accompany the introduction of a cross-strand disulfide and whether such structural changes could be correlated with the previously seen thermodynamic and catalytic changes. Solution structure studies were conducted using a suite of multidimensional heteronu-clear NMR experiments, for which isotopically enriched 15N and 13C, 15N labelled samples were used. The solution structure for 77c91cTrx was calculated using 1190 NOE distance restraints, 199 φ and ψ dihedral angle restraints and 48 hydrogen bond restraints. The solution structure for 78c90cTrx was calculated using 1123 NOE distance restraints, 197 φ and ψ dihedral angle restraints and 50 hydrogen bond restraints. The ensemble of structures for 77c91cTrx showed an RMSD of 0.78± 0.13 Å while the RMSD for the ensemble of structures of 78c90cTrx was seen to be 0.76±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were seen to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The tertiary structures of both proteins were seen to have a 5-strand mixed β-sheet and 4 helices surrounding it. . A comparison of the solution structures of mutant and wt -Trx showed significant changes in secondary and tertiary structure. For example, an α helix was reduced from 3 turns to a single turn, and of the β-strands containing the mutation was elongated by 3 residues. A ≈ 50% loss of hydrogen bonds, primarily from the β -sheet, was seen for both mutants. The secondary and tertiary structure for both 77c91cTrx and 78c90cTrx was seen to be near identical, despite the greater strain of the disulfide bond at the hydrogen bonding position. In addition to this, the Ile75-Pro76 peptide bond is now seen to be in the trans conformation in 78c90cTrx, while in wt -Trx the Ile75-Pro76 peptide bond is in the cis conformation. This cis peptide bond is known to play a role in both folding and catalysis, and the solution structures were analyzed in the context of observed changes in folding and catalysis. The study shows that introducing disulfide bonds even at the edge of β sheets have long-range structural effects, and the structural effects cannot be directly correlated with the changes in stability. Part III: Appendix Appendix A describes the expression, purification and preliminary characterization of ilvM, the regulatory subunit of E.coliAHAS II, one of three enzyme isomers that catal-yse the first step in the synthesis of all branched chain amino acids. AHAS II is known to be insensitive to feedback regulation, but our studies showed that the presence of Ile, Leu and Val causes structural changes and increases the stability of ilvM. However we were not able to purify ilvM in sufficient quantities to proceed with solution structure studies. Appendices B and C contain chemical shift information for the structural studies carried out on FFcytb5, WFcytb5, 77c91cTrx and 78c90cTrx.

Disulfides are the primary covalent interactions within a protein molecule that connect residues which are sequentially distant. Naturally occurring disulfides enhance the stability of the protein by destabilization of the unfolded state. Previous attempts to introduce disulfide bridges as a means to enhance protein stability have met with mixed results. Tools have been developed to predict potential sites for disulfide introduction. However, it must be noted that engineering disulfides is not a trivial task. The effect of the engineered disulfide on protein stability is difficult to predict. There have been few systematic studies carried out to study disulfides in the context of secondary structures. The work in this thesis is aimed at studying disulfides in two kinds of secondary structures- antiparallel β-sheets and helices. In particular, the focus in this thesis is on cross-strand disulfides in antiparallel β-sheets and intrahelical disulfides. The analysis of naturally occurring disulfides in these structural elements coupled with protein engineering studies in model proteins were used to understand the effects of introducing disulfides in helices and antiparallel β-sheets. Synopsis This thesis also includes studies carried out on molten globules of four periplasmic binding proteins of E.coli- Maltose binding protein (MBP), Leucine, isoleucine, valine binding protein (LIVBP), Leucine binding protein (LBP) and Ribose binding protein (RBP). Work carried out in the lab previously had shown that these molten globules can bind the ligands that the proteins do in their corresponding native states. The analysis of the thermodynamic data obtained for these molten globules by differential scanning calorimetry (DSC) studies and isothermal titration calorimetry (ITC) to characterize stability and ligand binding respectively are described in this thesis. To further study the structural features of molten globules by fluorescence resonance energy transfer (FRET), double cysteine mutants of MBP were constructed and characterized. The rationale behind the construction of these mutants and their characterization is reported. Chapter 1 gives an introduction to disulfides in proteins. Previous attempts at cataloguing and characterizing naturally occurring disulfides are described. An overview of studies carried out to determine the effects of removal of naturally occurring disulfides in proteins and the effect of engineered disulfides in different proteins is given. The various tools developed to predict potential disulfide sites are described. Chapter 1 also briefly discusses various aspects of molten globules and FRET. Chapters 2 and 3 involve studies with cross-strand disulfides occurring in antiparallel β-sheets. A detailed analysis on various stereochemical aspects of naturally occurring cross-strand disulfides is described in Chapter 2. The reasons for these disulfides to almost exclusively occur at non-hydrogen-bonded registered pairs have been explored with conformational analysis, modeling studies and energy calculations. In Chapter 3, the effect of engineering cross-strand disulfides in four model proteins- LBP, LIVBP, MBP and Top7 are described. The ease of formation of the introduced disulfides and their effects on protein stability are described. The proteins with engineered cross-strand disulfides at exposed positions were also examined for redox activity. Our studies have shown that in antiparallel strands, engineered disulfides at exposed NHB registered pairs provide a robust means of increasing protein stability. In Chapters 4 and 5, studies about intrahelical disulfides are described. In Chapter 4, the various conformational aspects of intrahelical disulfides occurring naturally are studied. Analysis of structures of proteins in conjunction with modeling studies show that all naturally occurring intrahelical residues bridge cysteines occurring between the N-Cap and 3rd residue of helices. To further explore conformational requirements for intrahelical disulfides, Cys pairs were introduced at N-terminal and interior of helices in a E.coli thioredoxin mutant lacking its active site disulfide. The ease of formation of the engineered disulfides, and their effects on protein stability were studied. The redox activity of the engineered disulfides was also examined. The studies demonstrated that intrahelical disulfides can only occur at the N-terminus of an α-helix and that the N-terminal CYS residue must adopt a non-helical backbone conformation. Although none of the engineered intrahelical disulfides increased the stability of the protein, they conferred mild redox activity. In Chapter 5, the ability of an engineered CXXC motif to bind Zn(II) is also explored. The effect of Zn(II) on the stability of the reduced and oxidized states of an engineered protein with a N-terminal intrahelical CXXC was ascertained. I have also shown that iminodiacetate (IDA) and nitrilotriacetate (NTA) resins charged with zinc can bind the protein CGPC 95-98 in reduced state. These Synopsis preliminary experiments on metal binding show that this property of CXXC motif could be exploited to develop a protein purification method. In Chapter 6, thermodynamic characterization of molten globules of four periplasmic binding proteins (LBP, LIVBP, MBP and RBP) is described. Studies had been previously carried out in the lab to characterize the stability and ligand binding of these molten globules. All four molten globules were found to bind their corresponding ligands without conversion to the native state. In Chapter 6, the estimation of ΔCp of unfolding and ligand binding from the DSC and ITC data is described. The binding of molten globules to their ligands and the ability to undego cooperative thermal unfolding indicated the presence of native protein-like tertiary contacts. To study the molten globule structure, we decided to construct double cysteine mutants of MBP for FRET studies. We decided to employ a strategy for differential labeling of the two cysteines with two different fluorophores based on the conformational differences between MBP in the ligand bound and free forms. Seven double cysteine mutants of MBP were made. The rationale behind the construction of these mutants and their preliminary characterization is described in the appendix to Chapter 6. The optimization of the differential labeling procedure of the MBP double mutants needs to be fine-tuned before further studies through FRET. The work described in this thesis has resulted in the following publications: 1.Prajapati RS, Indu S, Varadarajan R. Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins. Biochemistry. 2007 (46):10339-52. 1 Indu S, Kumar ST, Thakurela S, Gupta M, Bhaskara RM, Ramakrishnan C, Varadarajan R. Disulfide conformation and design at helix N-termini. Proteins.2010 (78):1228-42. 2 Indu S, Kochat V, Thakurela S, Ramakrishnan C, Varadarajan R. Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets. (Manuscript submitted)

碩士
國立臺灣大學
化學研究所
101
There are many factors that contribute to protein folding and structure stability: intrinsic propensity of amino acids, side chain ion pairing interaction, hydrophobic effect, hydrogen bonding, van der Waals interaction. In this study, we focused on the effect of lysine side chain length on sheet propensity at a non-hydrogen bonded strand position in β-hairpin. The β-hairpin peptides HPTAlaXaa (Xaa= Dap, Dab, Orn, Lys) were designed with the side chain of Lys9 systematically shortened to investigate the effect of Lys side chain length on sheet propensity. The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. All peptides were purified to 95% purity and were analyzed by 2D NMR experiments. Sequence specific assignment was performed. The hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants,and NOE signals.The fraction folded and ΔG of peptides were derived by comparing the chemical shifts with the fully folded and unfolded reference peptides. The percent folding of HPTAlaXaa peptides with Lys analogs at the guest position followed the trend: HPTAlaDap ~ HPTAlaDab < HPTAlaOrn ~ HPTAlaLys, showing that the longer the Lys analogue side chain, the more stable the β-haiprin structure. The HIV Rev protein binds RRE RNA to regulate the transport of unspliced and spliced mRNA from the nucleus to the cytoplasm posttranscriptionally. The Rev peptide is a random-coil. However, the conformation of the Rev peptide changes to an α-helix while binding to RRE RNA. Hydrogen bond surrogate (HBS) is one of the several cross-linking systems for stabilizing an α-helix, using the covalent bond C=C-C-N to substitute the C=O…H-N (i, i+4) hydrogen bond in a short helix. In order to synthesize an HBS peptide, strategy for synthesis of dipeptides that contained an allyl group on the amino group was designed and refined. Two wild type Rev peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. The secondary structure of the two peptides was random-coil analyzed by circular dichroism spectroscopy. The binding specificity of the Rev peptides was determined by gel shift assay. The dissociation constants of the Rev peptides were similar to previous studies.

Proteins performing catalytic roles predominantly occur in a few protein folds. Functional diversity within a common structural scaffold has been attributed to conformational features that enable exploration of reaction space. In this study, we examined specific aspects of functional diversity in the Double Stranded β-helix(cupin) fold. The cupin domain is a hyper-stable protein fold that can support a variety of functions. Variation in function using a conserved active site in the cupin fold is achieved by changes in the residues that line the active site cavity as well as by the choice of a metal cofactor. Although this appears to be a likely basis for functional diversification, a few exceptions exist. It is thus interesting to examine how enzymes with the same structure, metal cofactor and ligand coordination catalyze a diverse range of reactions. This thesis describes two bi-cupins, BacB (also known as bacilysin synthase, YwfC) and Quercetinase (YxaG). BacB is a part of the protein machinery involved in the synthesis of a di-peptide antibiotic bacilysin. The case of the bicupin protein BacB illustrates the problem of functional annotation of proteins with the cupin fold. None of the predicted functions for this enzyme could be experimentally validated in vitro. The crystal structure, determined by Single-wavelength Anomalous Dispersion (SAD) based on the bound metal-ion at the active site provided a basis to evaluate the catalytic role of this protein. Eventually, the function of this protein could be determined based on characterizing the gene product of bacA, the gene preceding bacB in the B. subtilis bac operon. The crystal structure determination of BacB also led to an analysis of multiple crystal forms, with implications for the role of molecular symmetry in forming protein crystals. The stability of the cupin domain was examined using B. subtilis quercetinase as a model system. The availability of the crystal structure and a robust activity assay enabled us to examine the role of fragment complementation in the stability of the cupin scaffold and its implications for the function of this enzyme. This thesis also has a section on the use of structural homology for function annotation for cupin proteins. The results presented here thus provide a frame-work to understand the structural basis for functional diversity in the cupin family. This thesis is organized as follows: Chapter 1: This chapter provides an introduction to the Double Stranded β-Helix-Helix (DSBH or cupin) fold. Proteins with a cupin scaffold are remarkably diverse - spanning both enzymatic and non-enzymatic functions. This chapter presents a compilation of previous reports encompassing eighteen different functional classes. These functions include seed storage, transcription factors and a host of various enzymatic activities. Cupin proteins can be monocupins, bicupins or multi-domain cupins based on the number of DSBH domains in a single polypeptide chain. Very few multi-domain cupin proteins have been identified and this is generally not considered to be a significant sub-group. The inference that cupin proteins with more than one domain are products of gene duplication events is also examined in detail. The latter part of this chapter aims to provide an introduction to the two model proteins B. subtilis BacB and Quercetinase. Chapter 2: This chapter describes studies on a bi-cupin protein BacB involved in bacilysin synthesis. Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. The production of this antibiotic is regulated via a stringent response and branches off the pathway for aromatic amino-acid biosynthesis at prephenate. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. This data, however, could not be reconciled with the putative functional assignments for these proteins whereby BacA, a prephenate hydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize L-anticapsin. Here, based on the characterization of the reaction products of BacA and BacB as well as the crystal structure of BacB, we demonstrate that B. subtilis BacB catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. The mass and NMR spectra of the reaction product of BacA suggest that BacA is a decarboxylase that acts on prephenate. BacB is an oxidase. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvicacid (PPY). A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis. Chapter 3 is based on the crystal packing analysis of three different crystal forms of B. subtilis BacB. BacB is an oxidase that catalyzes the production of the di-peptide antibiotic bacilysin. This protein is a bi-cupin with two double stranded β-helix domains fused in a compact arrangement. BacB crystallizes in three crystal forms, belonging to the triclinic, monoclinic and tetragonal space groups. These different crystal forms could be obtained in similar crystallization conditions. We also note that a slight disturbance to the crystallization droplet results in nucleation events, eventually resulting in a different crystal form. All three crystal forms of BacB diffract to high resolution, thus enabling the structure determination and analysis of the packing arrangements of BacB in different space groups. Metal ions at the lattice interface dominate the different packing arrangements. The crystal packing reveals that a dimer of BacB serves as the template on which higher order symmetrical arrangements are formed. BacB, however, is a monomer in solution. The different crystal forms of BacB thus provide experimental evidence to the hypothesis that molecular symmetry could aid crystallization. Chapter 4 provides a conformational analysis of the cupin fold using B. subtilis quercetinase as a model system to understand the conformational determinants of functional diversity. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this chapter thus show that the cupin scaffold offers a balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein. Chapter 5 is based on the phylogenetic analysis of cupin domains. The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary association and the nature of bound metal ion despite having a conserved β-barrel structural scaffold. Here, an attempt was made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which structures are available through world-wide structural genomics initiatives but characterized as “hypothetical”. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. This phylogenetic strategy was applied to BacB leading to clustering with oxidoreductases. BacB was experimentally demonstrated to be an oxidase. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises additional experimental details, methodology and aspects of the techniques used in this study. Appendix I contains a description of a methodology for Molecular Replacement (MR) calculations in obtaining phase information for protein crystallography. Appendix II provides additional details of experimental protocols.