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Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer
(2015)
The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.
Recently, two of the most common types of bone cancers in children and young adults have been proven to exhibit vulnerability to poly(ADP)-ribose polymerase, (PARP) inhibitors (e.g. olaparib, talazoparib). Ewing’s sarcoma (ES) are reported to harbor a fusion gene EWS-FLI1 (85%), inducing tumorigenesis. Additional, as the fusion gene acts as aberrant transcription factor, it similarly induces elevated PARP expression levels sensitizing ES to PARP inhibition. Second, by an exome sequencing approach in a set of primary osteosarcomas (OS) we identified mutation signatures being reminiscent of BRCA deficiency. Therefore, the sensitivity of a panel of OS cell lines to either talazoparib single treatment or in combination with several chemotherapeutic drugs was investigated.
To screen ES tumor cell lines against PARP inhibitors we applied four different PARP inhibitors (talazoparib, olaparib, niraparib and veliparib) that are frequently being used for clinical studies. We combined those PARP inhibitors with a set of chemotherapeutics (temozolomide (TMZ), SN-38, etoposide, ifosfamide, doxorubicin, vincristine and actinomycin D) that are part of the first-line therapy of ES patients. Here, we demonstrate how PARP inhibitors synergize with TMZ or SN-38 to induce apoptosis, whereas the combination of PARP inhibitors with the other drugs are not favorable. By investigation of key checkpoints in the molecular mechanisms of cell death, the pivotal role of the mitochondrial pathway of apoptosis mediating the synergy between olaparib and TMZ was revealed.
Employing talazoparib monotherapy in combination with or without several chemotherapeutic drugs (TMZ, SN-38, cisplatin, doxorubicin, methotrexate and etoposide/carboplatin), the correlation between homologous recombination (HR) repair deficiency (BRCAness) and the response to talazoparib as prototypical PARP inhibitor was validated in different OS cell lines. By calculation of combination indices (CI) and fraction affected (Fa) values, we identified TMZ as the most potent chemotherapeutic drug in combination with talazoparib inducing the mitochondrial apoptotic pathway in OS.
In our studies of two independent tumor entities with contrary genetic background we identified the combination of PARP inhibitor and TMZ as being most effective. Our studies point out that after TMZ induced DNA methylation and concomitant PARP trapping, DNA damage-imposed checkpoint kinase activation consequently induces G2-cell cycle arrest. Subsequent, PARP inhibitor/TMZ causes MCL-1 degradation, followed by activation of BAK and BAX, succeeding in loss of mitochondrial outer membrane potential (LMMP) and activation of downstream effector-caspases in mitochondrial apoptosis. Our findings emphasize the importance of PARP inhibition in order to chemosensitize ES, which express high PARP levels, or OS that bear features of BRCAness.
Functional dynamics of ribonucleic acids : development and application of spectroscopic tools
(2016)
Im Rahmen der vorliegenden Dissertation wird der Aufbau eines zeitaufgelösten Fluorimeters, die photophysikalische Grundcharakterisierung der drei 2-(Pyrenylethinyl)-Adenosine (PyAs) und das Wechselwirkungsgeflecht des tetracyclinbindenden Aptamers (TC-Aptamer) mit seinem Liganden Tetracycyclin (TC) und Mg2+ dargestellt.
Das zeitaufgelöste Fluorimeter basiert auf der experimentellen Technik des zeitkorrelierten Einzelphotonenzählens. Es verfügt über zwei Anregungsquellen: gepulste UV-LEDs und einen frequenzverdoppelten titandotierten Saphirlaser. Diese Quellen Decken einen Wellenlängenbereich von (310 - 550) nm ab. Das Spektrometer kann unter günstigen Umständen eine Zeitauflösung von 50 ps erreichen bei einer zeitlichen Messungenauigkeit von weniger als 0,02 %.
Die Leistungsfähigkeit des Aufbaus wird anhand einer umfangreichen Studie an den drei PyAs demonstriert.
Die drei PyAs 2-(1-Pyrenylethinyl)-Adenosine (1PyA), 2-(2-Pyrenylethinyl)-Adenosine (2PyA) und 2-(4-Pyrenylethinyl)-Adenosine (4PyA) sind eine Gruppe fluoreszierender RNA-Nukleosidanaloga, welche die Gesamtheit aller möglichen Konfigurations-isomere der Grundverbindung PyA umfassen. Ihre zeitabhängigen Fluoreszenzzerfallseigenschaften werden ergänzt von stationären Absorptions- und Fluoreszenzspektren, ultraschneller transienter Absorptionsspektroskopie und quantenchemischen Rechnungen. Die Fluoreszenz von 1PyA und 4PyA gehorchen der Regel von Kasha, wohingegen 2PyA einen triexponentiellen Zerfall mit ausgeprägter Abhängigkeit von der Anregungswellenlänge zeigt. Die transienten Absorptionsspektren aller drei Isomere weisen im gesamten Spektrum dominante, wenig strukturierte Absorptionsbanden des ersten angeregten Zustands auf, welche im nahen UV in unterschiedlichem Maße vom Grundzustandsbleichen und stimulierter Emission überlagert werden. 2PyA zeigt eine deutlich ausgeprägte Signatur für eine interne Umwandlung hin zum S1, wenn es in höhere angeregte Zustände angeregt wird.
Das Fluoreszenzverhalten von 2PyA wird mithilfe eines lokal angeregten (LE) und zweier intramolekularer Ladungstransferzustände, von denen einer der koplanaren Orientierung von Pyren und Adenin (MICT) und der andere einer um 90 ° verdrehten Orientierung (TICT) entspricht. Der LE-Zustand ist hierbei verknüpft mit dem S2 von 2PyA, welcher einer rein pyrenlokalisierten Anregung entspricht. Dieser Zustand existiert so in 1PyA und 4PyA nicht. Der verdrehte TICT-Zustand ist nur in 2PyA bevölkerbar, weil für 2PyA die Barriere zur Bildung von Rotameren am niedrigsten ist und das Molekül nach Anregung daher in diese Geometrie kommen kann und dann durch die stärkere elektrostatische Anziehung stabilisiert wird. 1PyA und 4PyA emittieren hingegen nur aus dem MICT-Zustand.
Die Komplexbildung des TC-Aptamers mit seinem Liganden TC in Lösung wird empfindlich beeinflusst durch die-Konzentration von Magnesiumkationen. Dies wird untersucht durch Bindungs- und Faltungs- und Denaturierungsstudien mit verschiedenen Mg- und Harnstoffkonzentrationen. Als experimentelle Observable dienen hierbei die konformationsabhängige Nukleobasenabsorption und ihr Zirkulardichroismus im fernen UV, die Fluoreszenz des Liganden TC und die freiwerdende Wärme der exothermen Bindungsreaktion des Aptamers mit Mg in An- und Abwesenheit von TC.
Ohne Mg ist eine Interaktion des TC-Aptamers mit TC nicht nachweisbar. Dies liegt daran, dass Mg die notwendige elektrostatische Abschirmung der negativen elektrischen Ladung am RNA-Rückgrat zur Verfügung stellt. Die Abschirmung erlaubt es dem Aptamer kompakte Strukturen mit tertiären Kontakten auszubilden. Wenn die Mg-Konzentration die Faltung des Aptamers vollständig unterstützt (> 1 mM), so befindet sich das Aptamer weitgehend in einer vorgefalteten Konformation, welche der bindungskompetenten stark ähnelt. In diesem Zustand kann das Aptamer seinen Liganden extrem schnell, nämlich annähernd diffusionslimitiert binden. Unter diesen Bedingungen hat TC kaum Einfluss auf die Konformation seines Aptamers.
Bei physiologischen Mg-Konzentrationen (0,2 - 0,8 mM) kann das Aptamer kompakte Konformationen mit tertiären Strukturen einnehmen. Diejenige Konformation, welche der bindenden sehr stark ähnelt, dominiert das konformationelle Gleichgewicht jedoch noch nicht vollständig, es ist lediglich eine Konformation von vielen möglichen. Daher eröffnen physiologische Mg-Konzentrationen dem TC-Aptamer Teile des Konformationsraumes, welche andernfalls nicht zugänglich wären und TC stabilisiert selektiv die native Konformation. Diese konformationelle Verschiebung liefert kann hierbei zur robusten Signalgebung für die Funktion als Riboschalter dienen.
This thesis deals with the NMR characterization of the structure and the folding dynamics of DNA G quadruplexes as potential therapeutic target in cancer therapy and building block for DNA based nanotechnology.
The first part of this thesis (Chapters 1-5) introduces the reader to the world of G quadruplexes.
The main features of the classic Watson Crick double helix and alternative non B DNA structures are illustrated in Chapter 1. Many different base pairing schemes are possible, besides the canonical Watson Crick motif, thereby expanding the structural complexity of DNA. Non canonical base pairing, such as Hoogsteen hydrogen bonding, enables the assembly of triplets and quartets, which are the building blocks of triplex and quadruplex structures, respectively.
The structural characteristics of DNA G quadruplexes are delineated in detail in Chapter 2.
G quadruplex structures are extremely polymorphic, in terms of strands orientation, loops geometry, grooves width and arrangement of the glycosidic torsion angles. The various structural elements as well as the different cation coordination geometries are here presented, with a special emphasis on the diversity of conformations reported for the telomeric DNA G quadruplexes.
Chapter 3 describes the biological roles of G quadruplex structures in the genome. After introducing the architecture of the telomeric DNA and its interacting proteins, the mechanism of the telomeres elongation catalysed by the telomerase enzyme and its implications for cancer are discussed. The occurrence of G quadruplex structures in functional regions of the genome, such as promoter regions of oncogenes, and their possible roles in regulating the gene transcription are then outlined in the second part of the chapter.
The potential of G quadruplex as a novel anti cancer target is examined in Chapter 4 and the proposed anti cancer mechanisms for a ligand stabilizing G quadruplex structures are discussed.
RNA G quadruplexes and their putative role in gene regulation at the level of translation are briefly illustrated at the end of the chapter.
A general overview on the NMR methods to investigate the G quadruplex structures is presented in Chapter 5. The experimental set up used for the real time NMR studies of the G quadruplex folding is also described.
The second part of the thesis (Chapters 6-8), which is the cumulative part, comprises the original publications grouped in three Chapters according to the topic.
The state of the art on small molecules targeting G quadruplex structures is given at the beginning of Chapter 6, including a summary of the experimental structures of G quadruplexes in complex with ligands available up to date. The publications presented in Chapters 6.1-6.3 are concerned with the elucidation of the interaction modes between DNA G quadruplexes and selected ligands with potential therapeutic applications.
The binding ability of two natural alkaloids (berberine and sanguinarine) to telomeric G quadruplexes is examined in Chapter 6.1. The ability of carbazole and diguanosine derivatives (synthetized in the group of Prof. Dash, IISER, Kolkata) to interact with c-MYC G quadruplex and down regulate c-MYC expression is explored in Chapter 6.2 and Chapter 6.3, respectively.
The energy landscape of human telomeric G quadruplex structures is discussed in Chapter 7, in light of the experimental kinetic studies as well as molecular dynamics simulations reported in literature until now. Up to date there is no general consensus regarding the folding pathway of unimolecular human telomeric G quadruplex, in particular due to the lack of atomic resolution data on the species involved in the folding. Chapter 7.1 presents the first real time NMR study of the human telomeric G quadruplex folding kinetics.
The final chapter of this thesis (Chapter 8) outlines the potential of G-quadruplex structures as building blocks in nanotechnology. After illustrating briefly the additional possibilities offered by alternative non B DNA structures to programme nanomaterials, a number of applications employing G quadruplex structures in different fields of nanotechnology are described. The article presented in Chapter 8.1 investigates the structural and photoswitching properties of a novel intermolecular azobenzene containing G quadruplex synthetized in the group of Prof. Heckel (Goethe University, Frankfurt).
Tectonin β-propeller containing protein 2 (TECPR2) was first identified in a mass- spectrometric approach as an interactor of GABARAP, an ATG8-family protein playing a role in autophagy. The mammalian ATG8 protein family consists of seven members, namely MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2. All share an ubiquitin-like core and possess two additional N-terminal α-helices, which are important for the distinct functions of the proteins. First determined in various organelles the ATG8 proteins are shown to be involved in autophagy, supporting the formation and cargo recruitment of autophagosomes, the vesicles transporting cargo for autophagic degradation.
Autophagy is the process of recycling cytoplasmic contents by degradation of misfolded proteins or damaged organelles in order to supply nutrients. Also clearance of pathogens can be achieved via autophagy. Importantly, LC3B is incorporated into the autophagosomal membrane and is therefore used as the main marker for autophagosomes. Previous studies exhibited that depletion of TECPR2 leads to a loss of LC3B-positive structures in cells, which suggests TECPR2 to positively regulate autophagic processes.
A frame shift deletion in the gene encoding for TECPR2 causes the generation of a premature stop codon and subsequent an unstable version of the protein, which is then degraded. Mutation in the TECPR2 gene triggers a neurodegenerative disorder termed hereditary spastic paraparesis (HSP). HSPs are a diverse group of neurodegenerative diseases that are characterized by spasticity in prevalent lower extremities and were mediated by a loss of axonal integrity of the corticospinal motor neurons. In the context of HSP more than 50 gene loci were identified by now. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy causing a form of HSP, the exact function of TECPR2 is unknown.
This study primarily focused on the determination of TECPR2’s binding mode to ATG8 proteins in vitro and in cells. The association of TECPR2 to all ATG8-family proteins was confirmed in in vitro pulldown experiments. Following fragment-based binding and peptide array experiments, the LC3-interacting region (LIR) of TECPR2 could be verified with mutants of TECPR2 lacking the LIR motif. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) were conducted to gain deeper insights into the binding preference to the different ATG8-family members. Moreover, the crystal structure of TECPR2-LIR was solved. In cells colocalization studies with overexpressed ATG8 proteins unraveled a preferential binding to the LC3-subfamily.
Further, mass spectrometric analysis revealed novel association partners of TECPR2: SEC24D, HOPS and BLOC-1, all of those participating in different endomembrane trafficking pathways. Interaction and colocalization of TECPR2 with these components was validated with several immunoprecipitation experiments and the N-terminal part of the protein comprising the WD40-domain could be defined as the binding site for all three of the association partners. In further approaches, the requirement of the LIR-motif and the necessity of the availability of LC3 protein for the particular interactions were determined. Interestingly, in the absence of LC3C the binding of TECPR2 to SEC24D was completely disrupted whereas a loss of LC3B only resulted in a decreased association. Notably, the binding proteins were not subjected to autophagosomal degradation, indicating that TECPR2 may operate as a multifunctional scaffold protein. While depletion of TECPR2 destabilized HOPS and BLOC-1, the autophagy defect observed in TECRP2-deficient cells could not be attributed to functional impairment of these two complexes.
Moreover, loss of TECPR2 led to a decline in protein levels of SEC24D and of its heterodimer partner SEC23A. Thus, TECPR2 is required to regulate the protein levels of SEC23A and SEC24D and subsequently the formation of the heterodimers. Together, SEC24D and SEC23A form the inner coat of COPII vesicles. These vesicles are responsible for the anterograde transport of cargo from the ER toward the Golgi compartment. COPII-coated vesicles are secreted form ER at distinct sites, termed ER exit sites (ERES). The small GTPase SAR1A maintains the vesicle budding, coating and secretion at the ERES. Together with SEC13, SEC31 forms the outer coat of the COPII vesicles and therefore serves as a general ERES marker.
Consistent with a defect in COPII coat assembly, the number of ERES diminished in the absence of TECPR2. These phenotypes could be rescued by the wildtype TECPR2 protein but not by the LIR-mutant. Intriguingly, these results were mimicked by depletion of LC3C, which localized to ERES. By monitoring the release of various cargos from ER in dependency of TECPR2 or LC3C, a role of both proteins in ER export was determined. These facts indicated that TECPR2 cooperates with LC3C to facilitate COPII assembly, ERES maintenance and ER export. Notably, fibroblast derived from a HSP patient carrying mutated TECPR2 showed diminished SEC24D protein levels and delayed ER export.
Concurrent with emerging evidence for a role of ERES in autophagosome formation, depletion of TECPR2 or LC3C or overexpression of a constitutive inactive SAR1 mutant reduced puncta formation of the early autophagosomal protein WIPI2.
In summary, this study uncovered a role for TECPR2 in ER export at ERES through interaction and stabilization of SEC24D, a COPII coat protein. This process also depended on ATG8-family protein LC3C, which is localized at ERES. Both proteins are required for correct COPII-mediated secretion. Moreover, the presence of TECPR2 and LC3C on ER allows development of omegasomes, membranous structures budding ER to form autophagosomes, by stabilization of WIPI2 and therefore contribute to autophagosome formation.
Transport processes across the membrane are essential to ensure survival of every living cell. Therefore, the exchange of membrane impermeable molecules is mediated by specific transport proteins, which are embedded in the lipid bilayer.
One important class comprises secondary active transporters, which couple very efficiently the uphill transport of the main substrate against its concentration gradient to the downhill transport of an additional substrate. These transporters are widely distributed among all kingdoms of life and accomplish many crucial functions. One function is to counteract the deleterious effect of hyperosmotic stress in bacteria. Several members of the BCCT (betaine-choline-carnitinetransport) family of secondary transporters mediate osmostress protection by the accumulation of the compatible solute betaine or its precursor choline (Lamark et al., 1991; Peter et al., 1996; Ziegler et al., 2010). Besides osmo-dependent sodium or proton-coupled symporters, the BCCT family includes few rare representatives of osmo-independent transporters such as the substrate:product antiporter CaiT from E. coli (Jung et al., 2002; Ziegler et al., 2010).
The best-characterized member of the BCCT family is the sodium-coupled betaine transporter BetP from Corynebacterium glutamicum. BetP together with the ABCtransporter OpuA and the H+-solute symporter ProP, became a paradigm for osmoregulated osmolyte transport. Although, all three transporters were extensively studied, the general mechanism of osmoregulation is still far from being understood. Thus, one task of this thesis was to elucidate further the regulatory properties of BetP.
BetP is tightly regulated by osmotic stress and is able to increase its basal betaine uptake activity dramatically upon elevated osmolalities within one second (Peter et al., 1998a). The osmotic stress is sensed by BetP via two stimuli, one is the increase of the internal K+ concentration above a threshold of 220 mM (Rübenhagen et al., 2001), the second is related to a change in the physical state of the membrane (Maximov et al., 2014). So far, several solved crystal structures in combination with functional and computational analysis provided insights into the coupling mechanism of betaine and its co-substrate sodium (Khafizov et al., 2012; Perez et al., 2012). Despite the wealth of data, the precise regulatory mechanism of trimeric BetP is still unclear.
Channelrhodopsin-2 (ChR2) is a light-gated cation selective channel from the unicellular alga Chlamydomonas reinhardtii, which is involved in phototaxis and photophobic responses. As other rhodopsins, ChR2 comprises a seven-transmembrane helix (TMH) motif and a retinal as the light-sensitive chromophore. The chromophore is covalently attached via a protonated Schiff base to the conserved lysine residue Lys257 located in TMH7. Based on its primary sequence and the all-trans configuration of the retinal in the ground state, ChR2 is assigned to the type I rhodopsins, also referred to as microbial-type rhodopsins. Upon light activation, the retinal isomerizes from the all-trans to the 13-cis form. This photoisomerization, which is accompanied by conformational changes of the protein, eventually leads to the opening of the channel and cation translocation. Cation flux during the conductive state leads to depolarization of the cell membrane and subsequent triggering of action potentials when expressed in neurons. Therefore, ChR2 has become the most versatile optogenetic tool, enabling a non-invasive investigation of neural circuits at high spatial and temporal resolution. With the rapidly increasing importance of ChR2 as a tool in neurobiology and cell biology, structural information is the prerequisite to an unambiguous understanding of the molecular mechanisms of this unique light-activated ion channel. The coupling between isomerization and structural alterations is well understood for other microbial-type rhodopsins, like bacteriorhodopsin (bR), halorhodopsin (HR) and sensory rhodopsin II (SRII). In case of ChR2, the first data on light-induced conformational changes came from spectroscopic studies and structural information is still missing. However, in order to fully understand the mechanism of light transduction by ChR2, it is necessary to determine the changes in the protein structure at specific steps in the photocycle.
By the time I started my PhD thesis, there was no structural information of ChR2 available. Therefore, the objective of this thesis was to obtain structural information of the transmembrane domain containing the first 315 amino acids of ChR2 by cryo electron crystallography. Besides revealing the structure of membrane proteins, cryo-EM of two-dimensional (2D) crystals is ideal for investigating conformational changes in membrane proteins induced by different stimuli. Therefore, the second objective of my thesis was the investigation of light-induced conformational changes in the slow C128T ChR2 mutant. The ~1,000 times longer lifetime of the open state of the C128T mutant compared to the wild-type allowed to trap different intermediates that accumulate during the photocycle.
In 2012, the X-ray structure of a channelrhodopsin-1/channelrhodopsin-2 chimaera (C1C2) at 2.3 Å resolution in the closed dark-adapted state was published (Kato et al., 2012). The structure revealed the essential molecular architecture of C1C2, including the retinal-binding pocket and the putative cation conduction pathway. Together with biochemical, spectroscopic, mutagenesis experiments, and the high-resolution model, some functionally important residues of ChR2 have been identified. However, unambiguous explanation of the molecular determinants that contribute to activation (gating) and transport were still mostly unknown.
RESULTS AND CONCLUSIONS
The first half of my theses dealt with 2D crystallization of ChR2. I succeeded in obtaining 2D crystals of ChR2 of four different types, which differed in size, crystal packing, crystal contacts and resolution, yielding structure factors up to 6 Å resolution. The crystals were grown by reconstituting the protein with different lipids at various lipid-to-protein ratios. The best crystals formed with the synthetic lipid DMPC and EPL upon detergent removal by dialysis. The projection maps calculated from these crystals revealed the overall structure of C128T ChR2 at 6 Å resolution and were published in 2011 (Müller et al., 2011). Surprisingly, ChR2 was found to be a dimer in all crystal types. The ChR2 dimer was stable both in detergent solution and in the presence of lipids for 2D crystallization. The monomers clearly showed the expected densities for the seven TMHs.
The arrangement of the ChR2 dimers on the four 2D lattices was different. However, comparison of the individual rojection maps revealed no significant differences within the ChR2 interface in the four crystal forms. The observation that the structure of the dimer was the same in all four crystal forms and in different lipids suggested strong specific contacts between the two protomers and implied that the protein was also dimeric in the native membrane. These findings were in agreement with Western blot analysis of plasma membranes from oocytes expressing ChR2 and laser-induced liquid bead ion desorption mass spectrometry, which both showed ChR2 as a dimer. The unusual stability of the ChR2 dimer contrasts with other microbial rhodopsins, which exist in different oligomeric states, i.e. monomers, trimers or dimers. These observations raised the question whether the functional unit is the monomer or the dimer.
The comparison of the projection map of the light-driven proton pump bR at the same resolution showed similar overall dimensions. Based on this comparison, the densities which became evident in the ChR2 projection maps could be assigned to the corresponding seven densities in bR. The shape of the densities near the dimer interface suggested that TMHs 2, 3, and 4 are oriented more or less perpendicular to the membrane plane, while the other four helices appear to be more tilted, as in bR.
Based on the high-resolution bR structure and the projection structures obtained, I have built a homology model. On the basis of this homology model, several residues found in the dimer interface were selected for mutational studies in order to disrupt the dimer interface.
The investigation of light-induced conformational changes in C128T ChR2 was the second part of my thesis. I designed an experimental setup for trapping light-induced conformational changes in C128T ChR2. In addition, I optimized the sample preparation in a way that the different illumination conditions did not alter the quality of the crystals. I have trapped two different functional states, namely the conductive open state and the non-conductive closed dark-adapted state.
In order to visualize the location and the extent of conformational changes, projection difference maps were calculated between the open and the closed state. Visual inspection of the difference maps between the open and the two closed states revealed three difference peaks that map to the TMHs 2, 6, and 7, indicating significant and specific rearrangements of these helices. The strong pair of positive/negative peaks at TMH6 suggests an outward tilt movement of approximately 2 Å. Close comparison of similar work on bR revealed that this movement is likely to occur at the cytoplasmic end of TMH6. A second highly significant negative peak is observed at TMH7, indicating a less pronounced tilt compared to TMH6. The third negative peak at TMH2 indicates a loss of density in this region. No significant differences were recorded at the TMH1, 5 and at the dimer interface formed by TMH3 and 4.
I succeeded in trapping and characterizing the open and closed state in the photocycle of ChR2 and could demonstrate that the transition from the closed to the open state is linked to significant light-induced tilt movements of TMH6 and 7, plus a loss of order in TMH2. These conformational changes are likely to create a large water-filled conducting pore, which seems to be required for the conductance of up to 2,000 ions per photocycle. The previously mentioned spectroscopic studies support the difference structures I obtained. This approach sets the stage for studying structural changes accompanying the formation and decay of other photocycle intermediates in ChR2. Future studies will aim at three-dimensional maps of the open and closed state at higher resolution.
The high selectivity of biological transformations taking place in Nature have long inspired synthetic chemists to develop analogous chemical processes. Similarly, transient intermediates identified in chemical transformations often provide a basis to understand biological processes. Therefore, new insights gained in biological studies are often useful for chemistry and vice versa.
Proteins, and catalytically active enzymes, are among the most essential units of living cells. Metalloproteins or -enzymes, i.e., proteins or enzymes that contain transition metal ions such as copper, nickel, iron or zinc are often involved in processes like (1) metal-ion storage and transport, (2) exchange of electrons with the environment in catalysis and electron transfer reactions, and (3) dioxygen storage, transport, and metabolization.
For decades, copper-mediated biological oxidations have spurred a great deal of interest among synthetic and catalytic chemists. Copper enzymes such as dopamine β-monooxygenase (DβM), peptidylglycine α-hydroxylating monooxygenase (PHM),particulate methane monooxygenase (pMMO) and tyrosinase activate molecular oxygen (O2) and incorporate one of the oxygen atoms selectively into C−H bonds yielding hydroxylated organic substrates. Remarkable progress in bioinorganic research has led to the development of a large number of copper-based model systems supported by various nitrogen donor ligands that bind O2, cleave the O−O bond, and/or afford hydroxylation reactions similar to copper enzymes. These synthetic model systems have helped to understand the structureactivity relationships of their biological role models and supporting theoretical studies have contributed substantially to the development of the field. Specifically, several density functional theory (DFT) studies have provided detailed mechanistic insights into coppermediated aliphatic and aromatic hydroxylation reactions. Until to date, however, pertinent quantum chemical research still suffers from severe problems as to identify sufficiently accurate and efficient methods for mechanistic studies, and conflicting literature reports have created confusions within the scientific community. Therefore, the first aim of this thesis is to identify a DFT method well suited to describe copper-mediated hydroxylation reactions. With this method at hand a number of interesting hydroxylation reactions is investigated aiming at a detailed understanding of the underlying reaction mechanisms.
The thesis is divided into four chapters of which the first, the introductory chapter, is further divided into three sections (1) copper proteins and enzymes, (2) copper-O2 reactivity in enzymes and (3) biomimetic Cu/O2 chemistry. The first section gives a brief overview of a number of copper enzymes. The second section provides a concise introduction to the biochemical transformations brought about by those copper enzymes that perform aliphatic and aromatic hydroxylation reactions. It is shown that such copper enzymes carry different types of active sites which are responsible for their specific biological functions. These copper enzymes with their biological function are the role models for synthetic chemistry. In the third section, biomimetic Cu/O2 chemistry, the insights gathered in the past 35 years of extensive research on copper-based synthetic model systems that mimic various aspects of copper-enzyme reactivity are reviewed. Various types of active copper sites have been realized in these synthetic model systems and a brief introduction to the respective reactivities towards C−H bonds is presented. We will specifically focus on isomerization processes of dinuclear active Cu2O2 sites and the specific reactivity aspects of these isomers, as these phenomena have been the subject of enormous research efforts aiming at the understanding of the function of the enzyme tyrosinase.
Theory has been integral part of this research and density functional theory (DFT) has effectively taken over the role as a working horse in most studies. Therefore, the second chapter is devoted to an exposition of earlier DFT applications in mechanistic studies of Cu/O2 chemistry. We specifically highlight the problems related to the use of DFT in this field and illustrate the present state of knowledge.
The third chapter of this thesis provides results and discussion of (1) DFT benchmark studies and (2) mechanistic studies. In the first section, the results of a careful benchmark study on the performance of various DFT methods to study the μ-η2:η2-peroxodicopper(II)/bis(μ-oxo)dicopper(III) core isomerization and the C–H hydroxylation processes are compared with available experimental reference data. We provide an assessment of the effects of relativity, counteranions, and dispersion on the reference reactions. The most suitable DFT method evolving from this study, BLYP-D/def2-TZVP including solvent and relativistic corrections, is applied in the next sections to investigate the mechanistic scenario underlying three copper-dioxygen mediated hydroxylation reactions of aliphatic and aromatic C–H bonds. Our mechanistic studies show that bis(μ-oxo)dicopper(III) complexes are capable of achieving selective aliphatic and aromatic C–H hydroxylations. The study of substituent effects in these reactions has further shown that the bis(μ-oxo)dicopper complex acts as an electrophile in hydroxylation.
The fourth chapter presents the conclusions of our investigations. Part of the work presented in this thesis has been published in a peer reviewed journal and enclosed in appendix 1. Further research work, not presented in chapters 1-4, was conducted during my PhD time. This has led to two publications which are added in the appendix.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
Cardiac progenitor cells hold great potential for regenerative therapies in heart disorders. However, the molecular mechanisms regulating cardiac progenitor cell expansion and differentiation remain poorly defined. Here we show that the multi- adaptor protein Ldb1, which mediates interactions between different classes of LIM domain transcription factors, is a multifunctional regulator of cardiac progenitor cell differentiation. Ldb1-deficient embryonic stem cells (ESCs) show a markedly decreased expression of second heart field (SHF) marker genes and subsequently impaired cardiomyocyte differentiation. Conditional ablation of Ldb1 in the early SHF using an Isl1-Cre driver led to embryonic lethality at Embryonic day (E)10.5 with cardiac abnormalities including a significantly smaller right ventricle and a shortened outflow tract, supporting a crucial role of Ldb1 in the SHF. Mechanistically we show that the importance of Ldb1 for SHF development is two-fold: On the one hand, Ldb1 binds to Isl1 and protects it from proteasomal degradation, as a consequence of which Ldb1-deficiency leads to an almost complete loss of Isl1+ cardiovascular progenitor cells. On the other hand the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing (3C- seq) identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. These interactions are of critical importance to regulate the expression of the downstream target genes since their expression levels are strongly dependent on the Ldb1/Isl1 levels. Overexpression of an Ldb1 mutant, which contains the LIM interaction domain and thereby can protect Isl1 protein from degradation, but lacks the dimerization domain and thus cannot promote long-range interactions, does not collaborate with Isl1 to regulate the expression of their common targets and results in defects in Isl1+ cardiac progenitor differentiation. In this thesis we show one of the first examples of genome-wide chromatin reorganization mediated by a developmental regulated, cell type specific, transcription complex. Ldb1 in concert with Isl1 promotes long range promoter- enhancer and enhancer-enhancer interactions in order to create active chromatin hub where gene important for heart development can be co-regulated. Moreover, Isl1 and Ldb1 genetically interact during heart development, as Isl1/Ldb1 haplodeficient embryos show various cardiac anomalies. The dosage-sensitive interdependence between Isl1 and Ldb1 in the expression of these key factors in cardiogenesis, further supports a key role of the Isl1/Ldb1 complex in coordinating a three dimensional genome organization, upstream of a regulatory network driving cardiac differentiation and heart development.
In conclusion, the Isl1/Ldb1 complex orchestrate a genome-wide three dimensional chromatin reorganization resulting in a transcriptional program responsible for the differentiation of multipotent cardiac progenitor cells into cardiomyocytes.
In der vorliegenden Arbeit galt es, stabile, lumineszente, tetrakoordinierte Organoborane unter Verwendung eines Bor-funktionalisierten ditopen Grundbausteins und unterschiedlicher π- konjugierter Ligandensysteme zu synthetisieren. Die Bifunktionalität sollte die gleichzeitige Einführung von zwei Lewis-Basen erlauben, um eine mögliche elektronische Kommunikation oder einen Energietransfer zwischen den Chromophoren zu gewährleisten.
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Zusammenfassend war es möglich unter Einsatz eines Bor-haltigen Grundsystems (DBA) durch die Variation der chelatisierenden bzw. verbrückenden π-konjugierten Liganden stabile und effiziente Fluorophore mit nützlichen optischen Eigenschaften zu realisieren.
Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.
During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine.
Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure.
In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions....
Die Biosynthese der Fettsäuren (FS) ist in Eukaryoten und Bakterien ein hochkonserviert zentraler Stoffwechselweg, der in zwei strukturell verschiedenen Systemen ausgeführt wird. Die meisten Bakterien, Parasiten, Pflanzen und Mitochondrien nutzen ein Fettsäuresesynthase Typ-II (FAS-II) System. Bei FAS II Systemen sind alle katalytischen Domänen separate lösliche Proteine. In Eukaryoten wie auch den Bakterien Corynebakteria, Mycobakteria, Nocardia (Klasse der CMN Bakterien) liegen die katalytischen Domänen fusioniert auf einer Polypeptidkette vor, die zu einem Multienzymkomplex der Fettsäuresynthase Typ I (FAS-I) assemblieren. Die Architektur der FAS-I zeigt große Unterschiede; die X förmige Säuger-FAS-I (Maier et al., 2006), sowie die fassartigen Enzyme der Pilz FAS-I (Jenni et al., 2007; Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008) und der bakteriellen FAS-I (Boehringer et al., 2013; Ciccarelli et al., 2013). Zwischen Pilz- und bakterieller FAS-I gibt es trotz des ähnlichen Aufbaus bedeutende Unterschiede. Mycobakterium tuberculosis, der Auslöser von Tuberkulose (TB), an der jährlich über eine Million Menschen weltweit sterben (WHO, 2014), synthetisiert durch eine Symbiose von FAS-I, FAS-II und der Polyketidsynthase-13 Mykolsäuren. Durch die Mykolsäuren ist M. tuberculosis resistent gegen äußere Einflüsse. FAS-I ist in die Synthese der Vorstufen der Mykolsäuren involviert. Sie stellt im Kampf gegen TB ein potentielles Inhibierungstarget dar.
Strukturell war die bakterielle FAS-I beim Beginn der vorliegenden Arbeit, nur durch negative-stain-Elektronenmikroskopie (EM) Aufnahmen aus dem Jahr 1982 charakterisiert (Morishima et al., 1982). In dieser Arbeit konnte die bakteriellen FAS I aus M. tuberculosis (MtFAS), sowie Corynebacterium ammoniagenes (CaFAS) und Corynebacterium efficiens (CeFAS) strukturell untersucht werden. Dies geschah mit den Methoden negative-stain-EM, Einzelmolekül-Cryo-EM (Cryo-EM), Cryo EM Tomographie (CET) und Röntgenkristallographie.
Anhand von CeFAS-Kristallen konnte erstmals durch Röntgenkristallographie die Struktur einer bakteriellen FAS-I bestimmt werden. Zudem wurde die hohe konformationelle Flexibilität der bakteriellen FAS-I mit mehreren Methoden gezeigt. Für die CaFAS konnte mit Cryo-EM initiale Prozesse der Proteinkristallbildung abgebildet werden.
A novel series of ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates was synthesized through the Kabachnik–Fields reaction using I2 as catalyst followed by copper-catalyzed cycloaddition of the azide–alkyne reaction (CuAAC). All structures of the newly prepared compounds were characterized by 1H NMR, 13C NMR, and HRMS spectra. The structures of 2e, 2f, 3d, and 3g were further confirmed by X-ray diffraction analysis. These compounds were tested against various strains of DNA and RNA viruses; compounds 4b and 4c showed a modest inhibitory activity against respiratory syncytial virus (RSV) and compound 4h displayed modest inhibitory activity against Coxsackie virus B4.
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
During my thesis, I worked on two different membrane proteins. One is a bacterial secondary transporter and the second is a human mitochondrial calcium channel.
The first part of my thesis involves the structural and biochemical characterization of an L-carnitine/ γ-butyrobetaine antiporter from bacteria called CaiT. The aim of the project was to understand the Na+ independence of CaiT and to determine the crystal structures of CaiT in different conformations to expand the mechanistic understanding of substrate/ product antiport in CaiT.
The study revealed how a positively charged amino acid side chain (arginine 262) in CaiT could structurally and functionally mimic a sodium ion. Additionally, various crystal structures of CaiT obtained in this study demonstrate that the central substrate-binding site is highly dynamic and can accommodate the substrate in various orientations.
In the second part of my thesis, I was able to optimize the expression and purification conditions for the human mitochondrial calcium uniporter or the MCU. Understanding how this channel functions can help us unravel the mechanism of calcium uptake by mitochondria. Secondary structure prediction analysis in combination with mass spectrometry of degraded MCU products obtained during the purification of the full-length protein led to the identification of a stable MCU construct. This study resulted in the successful purification of milligram quantities of stable MCU protein for the first time. Further optimization may be required to obtain more homogenous protein that is amenable for crystallization.
Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.