CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡ÃƒÂªE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.

No description available.

Cysteinyl peptides likely played an important role in the prebiotic synthesis of cofactors, such as iron-sulfur clusters. However, cysteinyl peptides must be reduced in order to coordinate iron-sulfur clusters. Mixtures of ferric ions and cysteinyl peptides leads to the reduction of ferric to ferrous ions and the concomitant formation of disulfide bridged, oxidized cysteinyl peptides that are incapable of coordinating an iron-sulfur cluster. Here, we develop a photochemically driven solution to this problem. Lipoic acid (( R )-5-(1,2-dithiolane-3-yl)pentanoic acid), a molecule structurally similar to fatty acids, can be photochemically reduced and can subsequently reduce the oxidized cysteinyl peptides needed for the coordination of an iron-sulfur cluster. Other dithilane ring containing molecules possess similar activity to lipoic acid. The synthesis of small peptides containing cysteines, such as glutathione and GCG (Gly-Cys-Gly) is easy by both solid phase and solution phase methodologies. However, as the length of the peptide increases, the yield begins to decrease, especially for peptides containing cysteines due to oxidation. One solution could be to exploit a previously uncovered mechanism for the joining of peptides into longer peptides. Such mechanisms, referred to as CPL for catalytic peptide ligation, rely on either thiols or metals as catalysts and peptide nitriles as substrates. Thus far, CPL has only been exploited with non-cysteinyl peptides. In this thesis, we extend CPL to cysteine containing peptides by taking advantage of the templating effects of Zn2+. Longer peptides with properly spaced cysteines are frequently better able to stabilize iron-sulfur clusters in aqueous solution than shorter peptides. Coordination can either be complete or an open coordination position, filled by solvent, can be used to bind substrate. Two well-known examples of such an arrangement are the radical SAM (S-adenosylmethionine) enzyme and aconitase being an enzyme of the citric acid cycle. We designed and synthesized peptide sequences that could coordinate a [4Fe-4S]2+ cluster with three cysteinyl ligands, leaving an open coordination position. The stability of the [4Fe-4S] cluster was affected by the intermediates of the citric acid cycle. The iron-sulfur can be reconstituted with the long peptidyl sequences from proteins such as SLC25A39 which contains four cysteine ligands to form [2Fe-2S] cluster, which is necessary for glutathione transport from cytosol to mitochondria.

Previous studies revealed that β-95-Cys provides an essential ligand to one of the Fe atoms on the P cluster within the MoFe protein of nitrogenase, and a limited number of substitutions at this position resulted in inactive nitrogenase. It was also found that the counterpart of β-95-Cys, α-88-Cys, which also acts as a cysteinyl ligand to the P cluster, is replaceable without a complete loss of activity. In order to study the structure-function relationship of the protein environment in this region with respect to the P-cluster, subtle changes were introduced at β-95-Cys in <i>Azotobacter vinelandii</I>nitrogenase through site-directed mutagenesis and gene replacement method. Some crude extracts from the mutants with substitutions at β-Cys contain typical FeMo cofactor EPR signal. The β-95^Asp MoFe protein also has significant nitrogenase activity, but lower, suggesting that β-Cys is not absolutely required for both FeMo cofactor insertion and nitrogenase activity. In order to characterize its catalytic features, the β-95^Asp MoFe protein was purified from mutant strain DJ1096. It has significantly reduced H⁺ reduction, C₂H₂-reduction and N₂-reduction activity. It was found that a higher percentage of electron flux goes to H⁺ compared to the wild type MoFe protein. It was also found that reductant independent ATP hydrolysis occurs during H⁺ reduction, suggesting that the altered MoFe protein has an increased affinity for Fe protein-ADP complex. Surprisingly, CO has a significant enhancement effect on H⁺ reduction at low electron flux, but not at high electron flux, and highly couples the electron transfer to ATP hydrolysis. These results indicate that the binding of CO to the MoFe protein may either decrease the affinity of Fe-ADP complex for the β-95^Asp MoFe protein or facilitate electron acceptance by the P cluster, thus improving the electron transfer to substrate. / Master of Science

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.

In der Kernspinresonanzspektroskopie (NMR) treten drei Effekte auf, die paramagnetische und diamagnetische Moleküle in isotroper Lösung unterscheiden: residuale dipolare Kopplung (RDC), Pseudokontaktverschiebung (PCS) und paramagnetische Relaxationsverstärkung (PRE). Alle drei Effekte sind abhängig von intermolekularen Winkeln und Abständen und können daher Informationen über die Struktur und Dynamik des Moleküls liefern. Um diese Informationen zu erhalten, muss das Molekül paramagnetische Eigenschaften aufweisen. Eine der heutzutage gebräuchlichen Methoden verwendet kleine molekulare Tags, die paramagnetische Metallionen koordinieren. Die meisten dieser Tags binden über eine Disulfidbrücke an Cysteine an der Proteinoberfläche. Um diese Methode für DNA anzuwenden werden daher neue Taggingstrategien benötigt. Im Rahmen dieser Arbeit wurde eine modifizierte Nukleobase synthetisiert, mit der ein Schwefelatom in die DNA eingebracht werden kann. Diese Methode erlaubt es, jeden Tag an die DNA zu binden, der als Verbindungsmethode eine Disulfidbrücke nutzt. Mit der Nukleobase wird eine Kohlenstoff-Dreifachbindung in die DNA eingefügt und mit Hilfe einer dipolaren Cycloaddition wird die freie Thiolgruppe eingebracht. Die modifizierte Nukleobase wurde erfolgreich an einem selbstkomplementären DNA-Strang (24 Nukleobasen) getestet. Die Nukleobase wurde während der Synthese der DNA eingefügt und der mit Lutetium, Terbium oder Thulium vorbeladene Cys-Ph-TAHA Tag wurde über eine Disulfidbrücke an die DNA gebunden. Die Beladung des Tags und die Taggingreaktion verliefen hierbei quantitativ. Nach diesem Erfolg war es ein Hauptaspekt dieser Arbeit, eine verlässliche und reproduzierbare Aufreinigungs- und Probenvorbereitungsmethode zu entwickeln. Diesem Punkt kommt besondere Bedeutung zu, da das Phosphatrückgrat der DNA, im Gegensatz zu Proteinen, Metallionen koordinieren kann. Im Theorieteil dieser Arbeit ist eine komplette Herleitung der drei Hauptmerkmale paramagnetischer NMR gegeben. Diese Herleitung beginnt bei Grundbegriffen des Magnetismus und neben den Gleichungen für RDCs, PCSs und PREs werden Ausdrücke für den dipolaren Hamiltonoperator, Kreuzrelaxationsraten, kreuzkorrelierte Relaxationsraten, durch Alignment induzierte RDCs, Korrelationsfunktionen und spektrale Dichten gegeben. Das zweite Thema dieser Arbeit basiert auf einem weiteren paramagnetischen Effekt. Um der reduzierten Empfindlichkeit der Kernspinresonanzspektroskopie verglichen mit anderen Spektroskopiemethoden entgegenzuwirken, wurden viele Methoden entwickelt, die auf eine Erhöhung der Polarisierung der Atomkerne zielen, d.h. um sogenannte hyperpolarisierte Kerne zu erzeugen. Eine dieser Methoden, die photochemisch erzeugte dynamische Kernpolarisierung (photo CIDNP), basiert auf kurzlebigen Radikalen, die durch direkte Laserbestrahlung der Probe im Magneten erzeugt werden. Im Rahmen dieser Arbeit wurde ein photo CIDNP Aufbau entworfen, gebaut und getestet. Die ersten Experimente und Resultate mit Triethylendiamin, L-Tyrosin und 3-Fluor-L-tyrosin zeigen die Vorteile und Grenzen dieser Methode auf. Für 3-Fluor-L-tyrosin wurde eine komplette Analyse des Relaxationsverhaltens, einschließlich der Kreuzrelaxation und der kreuzkorrelierten Relaxation, durchgeführt.

This thesis discusses the structure-function studies on triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf), directed towards understanding the roles of highly conserved residues by site derected mutagenesis. Chapter 1 provides an introductory overview to the relevant literature on triosephosphate isomerase. In addition, this Chapter provides an analysis of conserved residues in TIM, and amino acid diversity at specific positions in the structure using a dataset of 503 TIM sequences. Chapter 2 reports the work on the completely conserved residue, C126 in TIM, which is proximal to the active site. Five mutants, C126S, C126A, C126V, C126M and C126T have been characterized. Crystal structures of 3-phosphoglycolate (PGA) bound C126S mutant and the unliganded forms of the C126S and C126A mutants have been determined at a resolution of 1.7 Å to 2.1 Å. Kinetic studies reveal a ~5 fold drop in kcat for the C126S and C126A mutants, while a ~ 10 fold drop is observed for the other three mutants. All the mutants show reduced stability at lower concentration and higher temperature. Chapter 3 presents the kinetic and structural characterization for the E97Q and E97D mutants of Pf TIM. A 4000 fold reduction in kcat is observed for E97Q, 100 fold reduction for the E97D mutant, while a ~ 9000 fold drop in activity for the control mutant, E165A. A large conformational change for the critical K12 side chain is observed in the crystal structure of the E97Q mutant, while it remains unchanged in the E97D structure. The results are interpreted to invoke a direct role for E97 in the catalytic proton transfer cycle, eliminating the need to invoke the formation of the energetically unfavorable imidazolate anion at H95. Chapter 4 reports investigations with position 96 by the biochemical and structural characterization of single mutants, F96Y, F96A and the double mutants, F96S/S73A and F96S/L167V. F96Y showed ~100 fold drop in activity, F96A revealed ~10 fold drop in activity, while F96S/S73A showed 100 fold lower activity than that of the wild type enzyme. Interestingly, the double mutant F96S/L167V proved to be a partial pseudorevertant, showing 10 fold higher activity than the single mutant, F96S. Chapter 5 describes the cloning, and preliminary kinetic and biophysical characterization of the enzyme, Dm TIM. A survey of disease causing mutations in TIM and the relationship of these sites of mutation to the active site and the dimer interface of TIM is presented in this Chapter.

No description available.

Venomous animals have evolved a vast array of peptide toxins for prey capture and defense. Nature has evolved the venoms into a huge library of active molecules with high selectivity and affinity, which could be explored as therapeutics or serve as a template for drug design. The individual components of venom i.e. toxins are used in ion channel and receptor studies, drug discovery, and formulation of insecticides. ‘Venominformatics is a systematic bioinformatics approach in which classified, consolidated and cleaned venom data are stored into repositories and integrated with advanced bioinformatics tools and computational biology for the analysis of structure and function of toxins.’ Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors. Ziconotide (ω-conotoxin MVIIa from Conus magus (Magician's cone snail)), is proven as an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain (U.S. Food and Drug Administration. Center for Drug Evaluation and Research) attesting to the pharmaceutical importance of Conus peptides. From the point of view of protein sequence and structure analysis, conopeptides can serve as attractive systems for the studies in sequence comparison, pattern extraction, structure–function correlations, protein–protein interactions and evolutionary analysis. Despite their importance and extensive experimental investigations on them, they have been hardly explored through in silico methods. The present thesis is perhaps the first attempt at deploying a multi-pronged bioinformatics approaches for studies in the burgeoning field of conopeptides. In the process of sequence-structure-function studies of conopeptides, we have created several sequence patterns of different conopeptide families and these have been accepted for inclusion in international databases such as PROSITE, the first pattern database to have been developed (http://www.expasy.org/prosite) and INTERPRO (http://www.ebi.ac.uk/interpro). More importantly, we have carried out extensive literature survey on the peptides for which we have defined the patterns to create PROSITE compatible documentation files (PDOC6004, PDOC60025 and PDOC60027). We have also created a series of sequence patterns and associated documentation filesof pharmaceutically promising peptides from plants and venomous animals (including O-conotoxin and P-conotoxin superfamily members) with knottin scaffold. Knottins provide appealing scaffolds for protein engineering and drug design due to their small size, high structural stability, strong sequence tolerance and easy access to chemical synthesis. The sequence patterns and associated documentation files created by us should be useful in protein family classification and functional annotation. Even though patterns might be useful at the family level, they may not always be adequate at the superfamily level due to hypervariability of mature toxins. In order to overcome this problem, we have demonstrated the applicationos of multi-class support vector machines (MC-SVMs) for the successful in silico classification of the mature conotoxins into their superfamilies. TheI- and J-conotoxin-superfamily members were analyzed in greater detail. On the basis of in silico analysis, we have divided the 28 entries previously grouped as I-conotoxin superfamily in UniProtKB/Swiss-Prot (release 49.0) into I1 and I2 superfamilies inview of their having two different types of signal peptides and exhibiting distinct functions. A comparative study of the theoretically modeled structure of ViTx from Conus virgo, a typical member of I2-conotoxin superfamily, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels. Putative complexes created by us of very recently characterized J-superfamily conotoxin p11-4a with Kv1.6 suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. This finding provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profile against subtypes of the potassium channels for these conopeptides. Furthermore, the present thesis reports the application of order-statistic filters and hydrophobicity profiles for predicting the location of membrane-spanning helices. The Proposed method is in particular effective for the class of helical membrane proteins, namely the therapeutically important voltage-gated ion channels, which are natural targets of several conotoxins. Our suggested ab initio approach is comparatively better than other spatial filters, confirming to the efficacy of including the concept of order or ranking information for prediction of TM helicdes. Such approaches should be of value for improved prediction performance including in large-scale applications. In addition, anlaysis has been carried out of the role of context in the relationship between form and function for the true PDB hits of some nonCys-rich PROSITE patterns. We have found specific examples of true hits of some PROSITE patterns displaying structural plasticity by assuming significantly different local conformation, depending upon the context. The work was carried out as a part of the research interest in our group in studying structural and other features of protein sequence patterns. The Contributions of the candidate to venominormatics include, creation of protein sequence patterns and information highlighting the importance of the patterns as gleaned from the lteratures for family classification: profile HMM and MC-SVMs for conotoxin superfamily classification; in silico characterization of I1 and I2 conotoxin superfamilies; studies of interaction with Kv1 channels of typical members of I2 and 3 conotoxin superfamilies and development of improved methods for detecting membrane-spanning helices. Chapter I starts with a brief account of venominformatics; bioinformatics for venoms and toxins. Chapter 2 presents a regular expression based classification of Conus peptides. Chapter 3 revisits the 28 entries previously grouped as I-conotoxin superfamily in UniProt Swiss-Prot knowledgebase (release 49.0) having four disulfide bonds with Cys arrangement C-C-CC-CC-C-C and they inhibit or modify ion channels of nerve cells. Chapter 4 describes pseudo-amino acid composition and MC-SVMs approach for conotoxin superfamily classification. Chapter 5 describes in silico detection of binding mode with Kv1.6 channel of J-superfamily conotoxin p114a from bermivorouos cone snail, Conus planorbis. Chapter 6 presents a comparative sequence-structure-function analysis of naturally occurring Cys-rich peptides having the Knottin or inhibitor cystine knot(ICK) scaffold, from different plants and venomous animals based on information available in the knottin database(http://knottin.cbs.cnrs.fr/). Chapter 7 describes the application of order-statistic filters and hydrophobicity profiles for detecting membrane-spanning helices. Chapter 8 describes the role of context in the relationship between form and function for the true PDB hits of some non Cys-rich PROSITE patterns. Chapter 9 summaries the important findings of the present studies on naturally occurring bioactive Cys-rich peptides with emphasis on Conus peptides and their interactions with respective target such as voltage-gated ion channels.