The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

Introduction : L'hémophilie est une maladie génétique de la coagulation due à un déficit en facteur VIII ou en facteur IX. Ces déficits sont responsables d'un déficit du complexe ténase intrinsèque (VIIIa-IXa). De plus, le complexe ténase extrinsèque (facteur tissulaire - VIIa) est physiologiquement rapidement inhibé par le TFPI lié au facteur Xa. Nous avons évalué la capacité d'une forme tronquée du facteur Xa (GDXa), dépourvue de domaine Gla à se lier au TFPI et à soulager l'inhibition physiologique du complexe ténase extrinsèque. Matériel et Méthodes : Dans une première partie, nous avons évalué la capacité du GDXa à restaurer la génération de thrombine de plasmas d'hémophiles A et B sévères sans et avec inhibiteurs. Nous avons également comparé les profils de génération de thrombine obtenus après addition du GDXa à ceux obtenus en présence d'anticorps neutralisants anti-TFPI ou anti-antithrombine. Enfin, nous avons comparé les cinétiques enzymatiques de neutralisation du facteur Xa et du GDXa par le TFPI et l'antithrombine. Dans une seconde partie, nous avons étudié in silico les interactions entre la chaîne lourde du facteur Xa et le TFPI pour détecter les zones d'interaction défavorables. Cette étude a identifié des acides aminés du facteur Xa qui pourraient être substitués pour optimiser l'interaction avec le TFPI. Les résultats in silico ont orienté nos choix de mutagenèse dirigée pour concevoir différents variants moléculaires du GDXa (R138F, R138G, R138I) où l'arginine 138 est substituée. Ces variants protéiques ont été produits de façon recombinante dans des cellules HEK293E. La capacité des différents variants à restaurer la génération de thrombine de plasmas d'hémophiles a été testée avec les surnageants de culture cellulaires correspondants. Résultats : Dans la première partie, nous avons montré que le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles A et B sans et avec inhibiteurs. Comparativement au facteur Xa, le GDXa montre une affinité moindre pour le TFPI tandis que les affinités du GDXa et du facteur Xa pour l'antithrombine sont identiques. Enfin, malgré une demi-vie courte, l'effet du GDXa sur la génération de thrombine est maintenu pendant au moins une heure. Dans la seconde partie, nous avons produit les différents variants R138F, R138G et R138I en cellules HEK293E et montré que les surnageants de culture cellulaire étaient capables de restaurer la génération de thrombine de plasmas d'hémophiles de façon plus efficace que le GDXa. Conclusion : Comme le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles, nos résultats suggèrent que le GDXa pourrait être une alternative efficace aux thérapeutiques hémostatiques court-circuitantes actuelles chez les hémophiles sans ou avec inhibiteurs. Les résultats obtenus renforcent l'hypothèse que l'activité pro-coagulante du GDXa serait liée à la formation d'un complexe GDXa-TFPI limitant la formation du complexe Xa-TFPI nécessaire à l'inhibition physiologique du complexe ténase extrinsèque. De plus, notre approche rationnelle basée sur une étude in silico visant à augmenter l'affinité du TFPI pour le GDXa a permis de produire différents variants moléculaires du GDXa dont l'activité procoagulante in vitro est augmentée par rapport au GDXa. / Background: Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor (TFPI). We evaluated the ability of Gla-domainless factor Xa (GDXa), a truncated form of factor Xa devoid of procoagulant properties, to bind to TFPI and to alleviate the physiological inhibition of the extrinsic tenase. Design and Methods: In the first part of this work, we evaluated the ability of GDXa to restore coagulation in plasmas from hemophilia A and B patients without and with inhibitors, using a thrombin generation assay triggered by a low concentration of tissue factor. We then compared its efficacy to generate thrombin to depletion of antithrombin or TFPI by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and GDXa by antithrombin and TFPI. In the second part of this work, we realized an in silico study of the interactions between factor Xa heavy chain and TFPI. The aim was to detect unfavorable interactions and to identify amino-acid candidates for mutagenesis in order to increase affinity for TFPI. Taking into account the results of this in silico study, we produced by genic engienering different molecular variants of GDXa (R138F, R138G, R138I) where Arg138 was substituted by site directed mutagenesis. Proteins were produced in HEK293E cells. We tested dialyzed cell culture supernatants containing each variant to restore thrombin generation in plasmas from severe hemophilia patients. Results: In the first part of this work, we showed that GDXa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. GDXa had a lower affinity than factor Xa for TFPI whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of GDXa on thrombin generation was sustained for at least one hour. In the second part of this work, we produced the different variants R138F, R138G et R138I in HEK293E cells and showed that cell culture supernatants were able to restore thrombin generation in a more efficient way than GDXa. Conclusions: As GDXa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without inhibitors. Results sustained the hypothesis that GDXa coagulant activity is through TFPI binding and competition with factor Xa to bind TFPI resulting in limiting factor Xa-TFPI formation, which is essential for inhibition of extrinsic tenase complex. Furthermore, rational design of GDXa variants based on an in silico study lead to production of proteins whose coagulant activity is increased compared to GDXa."

Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...

A troponina (Tn) regula a contração do músculo estriado esquelético de vertebrados. Ela é composta de três subunidades: troponina I (TnI), troponina C (TnC) e troponina T (TnT). A TnI tem a função inibitória que é neutralizada pela ligação de Ca2+ nos sítios regulatórios do N-domínio da TnC, e a TnT posiciona o complexo no filamento fino. Para monitorar o sinal do Ca2+ sendo transmitido da TnC para a TnI as propriedades espectrais únicas do 5-hidroxitriptofano (5HW) foram utilizadas. O 5HW foi incorporado em mutantes pontuais de TnI com um único códon para triptofano. Foram identificadas duas sondas espectrais intrínsecas na TnI capazes de detectar a ligação de Ca2+ na Tn: as TnIs com 5HW nas posições 100 e 121. Complexos troponina reconstituídos com estes mutantes fluorescentes de TnI, Tn-TnIF100HW e Tn-TnIM121HW, apresentaram respectivamente 12 e 70 % de aumento na intensidade do espectro de emissão devido à ligação de Ca2+ na TnC. Nos complexos binários (TnC-TnI) as TnIs com 5HW nas posições 106 e 121 também captam a ligação do Ca2+ na TnC. A análise da fluorescência destas sondas demonstrou que: 1) as regiões da TnI que respondem ao N-domínio regulatório da TnC ocupado com Ca2+ são a região inibitória da TnI, resíduos 96 até 116, e a região vizinha que inclui a posição 121 da TnI; 2) mutações pontuais e a incorporação de 5HW na TnI podem afetar tanto a afinidade como a cooperatividade da ligação de Ca2+ na TnC, confirmando o papel da TnI em modular a afinidade da TnC por Ca2+; 3) as constantes de dissociação de Ca2+ surpreendentemente altas, Kd ~ 10-8 M, calculadas a partir dos sinais das sondas na região inibitória da TnI, sugerem a possibilidade de que os sítios do domínio N-terminal da TnC sejam os sítios de ligação de Ca2+ de maior afinidade no complexo troponina. / Vertebrate striated muscle contraction is regulated by troponin (Tn). Tn is composed of three subunits: troponin I (TnI), troponin C (TnC) and troponin T (TnT). TnI has an inhibitory role that is neutralized by calcium binding to the regulatory sites in the N-domain of TnC, and TnT positions the troponin complex on the thin filament. In order to follow the Ca2+ induced conformational change that is transmitted from TnC to TnI, the unique spectral properties of 5-hydroxytryptophan (5HW) incorporated as point-mutants of TnI were used. It was possible to identify two new TnI intrinsic spectral probes sensitive to Ca2+ binding to Tn: TnI with single 5HW at positions 100 and 121. Trimeric troponin complexes reconstituted with two fluorescent mutants of TnI, Tn-TnIF100HW and Tn-TnIM121HW, showed respectively 12 and 70 % increase in the emission spectra when Ca2+ bound to TnC. In the binary complexes (TnC-TnI) two TnIs with 5HW at positions 106 and 121 were also sensitive to Ca2+ binding to TnC. Fluorescence analysis of these probes showed: 1) the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region of TnI (residues 96 to 116), and a neighbor region that includes position 121; 2) point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC, confirming the role of TnI as a modulator of the Ca2+ affinity of TnC; 3) the high dissociation constant for sites in the N-terminal domain of TnC (Kd ~ 10-8 M), derived from data using probes in the inhibitory region of TnI suggested the possibility that these sites are the high affinity Ca2+ binding sites in the troponin complex.

DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

A troponina I (TnI) é a sub-unidade inibitória do complexo troponina, responsável pela regulação da contração do músculo esquelético. Foi demonstrado que sua ação inibitória sobre a Mg2+ATPase da actomiosina, deve-se principalmente à região entre os resíduos 96 e 116 (região do peptídeo inibitório). Para estudar o mecanismo de inibição a nível molecular, produzimos três mutantes na região do peptídeo inibitório através de mutações sítio-dirigidas. Substituímos os resíduos lisina 105 por ácido glutâmico (K105E), fenilalanina 106 por tirosina (F106Y) e arginina 113 por ácido glutâmico (R113E). As troponinas I mutantes foram expressas em E.coli, purificadas e ensaiadas em sua atividade inibitória, interações com os outros componentes do complexo regulatório e sua capacidade regulatória. Os resultados obtidos indicam que a mutação na posição 105 alterou a interação da proteína com a tropomiosina, diminuindo sua atividade inibitória e afinidade pela actina-tropomiosina. A substituição na posição 113 alterou a interação da proteína com a actina e com a actina-tropomiosina, também diminuindo a atividade inibitória na presença de tropomiosina e inviabilizando a inibição na ausência de tropomiosina. Já a substituição na posição 106 não produziu alteração detectável. Concluímos que o resíduo 105 faz parte do sítio de ligação da troponina I ao complexo actina-tropomiosina e que o resíduo 113 participa diretamente do mecanismo de inibição. Desta forma, definimos duas interfaces de interação da troponina I com o filamento de actina-tropomiosina, necessárias a ligação da troponina I ao filamento e inibição da ATPase. / Troponin I (TnI) is the inhibitory subunit of the troponin complex, responsible for the regulation of skeletal muscle contraction. It has been demonstrated that TnI\'s inhibitory action on Mg2+ATPase of actomyosin is due principally to the region between residues 96 and 116 (the inhibitory region). To study the inhibitory mechanism at the molecular level, we produced three mutants of the inhibitory region by site-directed mutagenesis. We substituted lysine 105 for glutamic acid (K105E), phenylalanine 106 for tyrosine (F106Y) and arginine 113 for glutamic acid (R113E). The TnI mutants were expressed in E. coli, purified and analyzed for their inhibitory activity, interaction with other components of the regulatory complex and regulatory capacity. The results indicate that the mutation in K105E modified the interaction of TnI with tropomyosin, reduced its inhibitory activity and actin-tropomyosin affinity. The mutant R113E displayed modified interaction with actin and actin-tropomyosin, reduced inhibitory activity in the presence of tropomyosin and essentially no inhibitory activity in the absence of tropomyosin. The mutant F106Y behaved essentially like wild-type TnI. We conclude that residue 105 is part of the site by which troponin I binds to the actin-tropomyosin and that residue 113 participates directly in the inhibitory mechanism. In this way, we have defined two interfaces between troponin I and the actin-tropomyosin which are necessary for binding TnI to the filament and to inhibit the actomyosin ATPase.

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multispecific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.

The cardiac sodium ion channel (NaV1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na+ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits NaV1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on NaV1.5, we perform docking calculations and high-throughput electrophysiology experiments in the present study. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of NaV1.4 and NaV1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at NaV1.5