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In mitochondrial respiration, the soluble protein cytochrome c accepts an electron from the membrane bound cytochrome bc1. The interaction between cytochrome bc1 and cytochrome c is highly transient in nature, enabling turnover numbers greater than 160 s-1. Yeast cytochrome bc1 has been successfully crystallised with bound cytochrome c with the help of an antibody fragment (Lange and Hunte 2002; Solmaz and Hunte 2008). In all crystal structures of the complex, the homodimeric cytochrome bc1 binds only one cytochrome c, with the binding site located on subunit cytochrome c1. Univalent cytochrome c binding is correlated with conformational changes of the Rieske protein head domain and subunit QCR6p. The interface of the complex is small. The haem moieties are centrally located in a mainly non-polar contact site that includes a cation–! interaction and is surrounded by complementary charged residues. The crystal structure is in agreement with the general architecture of the interfaces of transient redox complexes and also reveals several interesting features unique to the cytochrome bc1. On the basis of the crystal structures, an extensive thermodynamic and kinetic characterisation of the interaction was carried out in this work to challenge the static snapshot of the bound proteins in the crystal structure as the relevant physiological electron transfer. The thermodynamic parameters of the interaction between the redox partners were determined using isothermal titration calorimetry (ITC). The association constant for cytochrome bc1 and cytochrome c in oxidised state under physiological ionic strength of 120 mM at 25 °C, was determined to be 5 " 103 M-1 by direct ITC titration. So, the partners interact with an affinity of 200 #M. In spite of the low affinity the complex has a life time ($ = 1/koff) of 5 #second, sufficiently long to enable the theoretically calculated electron transfer rates of 1.0 " 106 to 2.6 " 107 s%1 with a lifetime ($ = 1/rate) of 1-0.04 μseconds and experimentally determined rate of 7.7 " 104 s%1 with a lifetime of 13 μseconds. The low affinity makes it difficult to ascertain the stoichiometry of binding. The enthalpy of the interaction is endothermic, which is consistent with the nature of an interface where hydrophobic interactions are dominant. The enthalpy and entropy is 3.6 kJmol-1 and 83 kJmol-1K-1, respectively. The importance of key interface residues was also investigated. The role of the interface residue G89 of cytochrome c which might have a role in the dissociation of the complex has been probed by site-directed mutagenesis. The interface contains a cation-! interaction between F230 of cytochrome bc1 and R19 of cytochrome c, which is thought to provide the specificity to the interaction between the otherwise promiscuous partners. To analyse the role of this interaction pair in electron transfer, F230L and F230W mutants were used to measure direct electron transfer rates by flash photolysis and steady state kinetics. The findings indicate that another ! system can work as functional substitution of F230, while deleting the ! system has a deleterious effect on the complex formation. The inability of F230L to achieve the transient and steady state turnover rates as wild type protein indicates a scenario where the variant achieves an altered bound state with inefficient electron transfer pathways and higher edge-to-edge distance. The role of supernumerary subunit QCR6p in complex formation was investigated by steady state kinetics measurements. Subunit QCR6p does not interact directly with cytochrome c but is positioned in such a way that it could electrostatically steer cytochrome c in a reactive ensemble. The highly acidic and disordered N-terminus of QCR6p could interact with a patch of conserved lysine residues on cytochrome c. The role of subunit QCR6p has been assessed using QCR6p deleted cytochrome bc1 and a lysine variant of cytochrome c. The results show that QCR6p not only affects the kinetics of the interaction but is also important for the stability of cytochrome bc1. The kinetic and thermodynamic data obtained during this study provide evidence for the functional importance of non-catalytic cytochrome bc1 subunit QCR6p, show that the entropy driven interaction is indeed of low affinity and highly transient in nature and indicate that the interface is well suited to ensure the high turnover of the electron transfer chain where cytochrome c interacts with multiple partners using overlapping interfaces. The suggested role of the cation-! interaction as a highly specific interaction has been validated.
In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, reduces its kinase activity and increases the sensitivity for Imatinib. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; iii.) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.
The various OPE mixtures were also tested on sSOI material which consists of a thin strained silicon layer on top of an insulator like silicon dioxide. The OPE A, B and F are able to reveal threading dislocations (TD) in the strained silicon film (chapter 5.11). The TD densities determined for the OPE A correspond very well with those obtained with the Secco diluted reference. The tested OPE mixtures are not able to delineate other crystal defects like stacking faults, pile ups or twins, which also appear in the strained silicon. Some Organic Peracid Etches were also tested on wafers with an epitaxial silicon layer and on silicon substrates. Epitaxially produced silicon layers are nearly defect-free. Etching times were chosen such that only a part of the epitaxial layer was removed. Nevertheless, after very long etching times (> 16 h) isolated pits were found, with defect densities ranging from 104/cm3 to 106/cm3 depending on the etching solution used. No etch pits were found in the remaining epitaxial layer when OPE F was used. Longer etching times appear to favour the formation of artefects. These artefacts could be caused by the formation of gas bubbles, particles or micro scratches at the crystal surface. The OPE C and D are able to reveal vacancy agglomerates (D-defects) in silicon substrates (see under 5.5, 5.6 and 5.11in chapter 5). Due to their low removal rates and the long etching times which favour the formation of artifacts, these solutions are less suited to the delineation of defects in silicon substrates. In the second part of this study the different etch formulations have been compared with each other in respect of their physical properties like removal rates, activation energies, standard potentials and selectivities (chapter 6). The selectivity was determined at etch pits caused by dislocations. The depth of the etch pits, determined by atomic force microscopy (AFM), should be dependent on the selectivity of the corresponding etching solution used. The higher the selectivity of the solution the deeper the etch pit should be. It was assumed that a low removal rate and a high activation energy for the etching process should correspond to a high selectivity. However, the experimental results have shown that it is not possible to predict the selectivity of an etching solution from experimental parameters like removal rate or activation energy. One must bear in mind that selectivity was only determined on one particular type of crystal defect, namely on dislocations. Values for selectivity in the etching solutions can differ for other defect types. Besides the etching solutions used in this study differ considerably from each other in respect of their chemical and physical roperties. They can be divided into three completely different etching systems. The original Secco solution and the diluted variations thereof are hydrofluoric acid-dichromate mixtures with the Cr6+ species as the oxidizing agent. The Jeita and MEMC solutions contain nitric acid, hydrofluoric acid and, as diluents, acetic acid and water. Here the oxidizing agents are various N(III) species which are formed autocatalytically during the etching process. The concentration of acetic acid also plays an important role as it lowers the degree of dissociation of HF and of HNO3. This has an influence on the pH and the standard potential of the etching solution. The Organic Peracid Etches are mixtures of hydrogen peroxide and a short-chain alkanoic acid like acetic acid. Such systems are strictly speaking not aqueous solutions, the reactive species is the peracid formed.Within each system, however, a certain relationship is perceived between the selectivity of the etching solution on the one hand, and the and the activation energy or the removal rate on the other. The decreased activation energy for the etching process of silicon at a dislocation can be calculated from experimental data by using the Arrhenius equation (chapter 6.3). It was found that the strain inside the crystal lattice caused by a dislocation loop leads to an increase of the potential energy of ~ 5 % and, hence, a decrease of the activation energy of ~ 5 % and an increase in the removal rate of ~ 100 %.
Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
1. Fab co-complexes of proton pumping NADH:ubiquinone oxidoreductase (complex I) Fab fragments suitable for co-crystallization with complex I were generated using an immobilized papainbased protocol. The binding of the antibody fragments to complex I was verified using Surface Plasmon Resonance and size exclusion chromatography. The binding constants of the antibodies and their respective Fab fragments were found to be in the nanomolar range. This work presents the first report on successful crystallization of complex I (proton pumping NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica with proteolytic Fab fragments. The quality of the crystals was significantly improved when compared to the initial experiments and the best crystals diffracted X-rays to a resolution of ~7 Å. The activity of complex I remained uninfluenced by antibody fragment binding. The initial diffraction data suggest that the complex I/Fab co-complex crystals represent a space group different to the one observed for the native protein. Ongoing experiments are aimed at further enhancements of the diffraction quality of the crystals. Providing a different space group the CI/Fab co-complexes may become a very useful approach for structure determination of the enzyme. Moreover, the bound Fab offers an additional possibility to generate phase information. The antibody-mediated crystallization represents a valuable tool in structural characterization of the NADH:oxidoreductase subcomplexes or even single subunits. 2. UDP-glucose pyrophosphorylase UDP-glucose pyrophosphorylase from Yarrowia lipolytica displays affinity towards Ni2+ NTA and was first detected in a contaminated sample of complex I. Following, separation from complex I, Ugp1p was purified using anion exchange chromatography. Sequence similarity studies revealed high identity to other known pyrophosphorylases. As indicated by laser-based mass spectrometry method (LILBID) Ugp1p from Y. lipolytica builds octamers similarly to the enzyme from Saccharomyces cerevisiae. The initial crystals grew as thin needles favorably in sitting drop setups. The size of the crystals was increased by employment of a micro batch technique. The improved crystals diffracted X-rays to a resolution of 3.2 Å at the synchrotron beamline. Structural characterization is under way using a molecular replacement approach based on the published structure of baker’s yeast UGPase.
By adopting a variety of shapes, proteins can perform a wide number of functions in the cell, from being structural elements or enabling communication with the environment to performing complex enzymatic reactions needed to sustain metabolism. The number of proteins in the cell is limited by the number of genes encoding them. However, several mechanisms exist to increase the overall number of protein functions. One of them are post-translational modifications, i.e. covalent attachment of various molecules onto proteins. Ubiquitin was the first protein to be found to modify other proteins, and, faithful to its evocative name, it is involved in nearly all the activities of a cell. Ubiquitylation of proteins was believed for a long time only to be responsible for proteasomal degradation of modified proteins. However, with the discovery of various types of ubiquitylation, such as mono-, multiple- or poly-ubiquitylation, new functions of this post-translational modification emerged. Mono-ubiquitylation has been implicated in endocytosis, chromatin remodelling and DNA repair, while poly-ubiquitylation influences the half-life of proteins or modulates signal transduction pathways. DNA damage repair and tolerance are example of pathways extensively regulated by ubiquitylation. PCNA, a protein involved in nearly all types of DNA transaction, can undergo both mono- and poly-ubiquitylation. These modifications are believed to change the spectrum of proteins that interact with PCNA. Monoubiquitylation of PCNA is induced by stalling of replication forks when replicative polymerases (pols) encounter an obstacle, such as DNA damage or tight DNA-protein complexes. It is believed that monoubiquitylation of PCNA stimulates the exchange between replicative pols to one of polymerases that can synthesize DNA across various lesions, a mechanism of damage tolerance known as translesion synthesis (TLS). Our work has helped to understand why monoubiqutylation of PCNA favours this polymerase switch. We have identified two novel domains with the ability to bind Ub non-covalently. These domains are present in all the members of Y polymerases performing TLS, and were named Ub-binding zinc finger (UBZ) (in polη and polκ) and Ub-binding motif (UBM) (in polι and Rev1). We have shown that these domains enable Y polymerases to preferentially gain access to PCNA upon stalling of replication, when the action of translesion polymerases is required. While the region of direct interaction between Y pols and PCNA had been known (BRCT domain in Rev1 and PIP box motif (PIP) in three others members), we propose that Ub-binding domains (UBDs) in translesion Y pols enhance the PIP- or BRCT-domain-mediated interaction between these polymerases and PCNA by binding to the Ub moiety attached onto PCNA. Following these initial studies, we have also discovered that Y polymerases themselves undergo monoubiquitylation and that their UBDs mediate this modification. This auto-ubiquitylation is believed to lead to an intramolecular interaction between UBD and Ub attached in cis onto the UBD-containing protein. We have mapped monoubiquitylation sites in polη in the C-terminal portion of the protein containing the nuclear localization signal (NLS) and the PIP box. Beside PIP, the NLS motif is also involved in direct interaction of polη with PCNA. Based on these findings, we propose that monoubiquitylation of either NLS or PIP masks them from potential interaction with PCNA. Lastly, using several functional assays, we have demonstrated the importance of all these three motifs in the C-terminus of polη (UBZ, NLS and PIP) for efficient TLS. We have also constructed a mimic of monoubiquitylated polη by genetically fusing polη with Ub. Interestingly, this chimera is deficient in TLS as compared to the wild-type protein. Altogether, these studies demonstrate that the C-terminus of polη constitutes a regulatory module involved in multiple-site interaction with monoubiquitylated PCNA, and that monoubiquitylation of this region inhibits the interaction between polη and PCNA. Our work has also revealed that the UBDs of Y pols as well as of other proteins implicated in DNA damage repair and tolerance, such as the Werner helicase-interacting protein 1 (Wrnip1), are required for their proper sub-nuclear localization. All these proteins localize to discrete focal structures inside the nucleus and mutation of their UBDs results in inability to accumulate in these foci. Interestingly, by exchanging UBDs between different proteins we have learned that each UBD seems to have a distinct functional role, surprisingly not limited to Ubbinding ability. In fact, swapping the UBZ of Wrnip1 with the UBM of polι abolished the localization of Wrnip1 to foci despite preserving the Ub-binding ability of the chimeric protein. In summary, this work provides an overview of how post-translation modification of proteins by Ub can regulate several DNA transactions. Firstly, key regulators (e.g. PCNA) can be differentially modified by Ub. Secondly, specialized UBDs (e.g. UBM, UBZ) embedded only in a subset of proteins act as modules able to recognize these modifications. Thirdly, by means of mediating auto-ubiquitylation, UBDs can modulate the behaviour of host proteins by allowing for either in cis or in trans Ub-UBD interactions.
Enantioselective carbon-carbon bond-forming reactions, particularly, using organocatalysts represent one of the most important areas in modern synthetic chemistry. New concepts and methods in organocatalysis are emerging continuously, allowing more selective, economically more appealing and environmentally friendlier transformations. Chiral Brønsted-acid catalysts have recently emerged as a new class of organocatalysts for a number of enantioselective carbon-carbon bond-forming reactions. The first part of this thesis focused on the new development of new Brønsted acid-catalyzed enantioselective Nazarov cyclizations. The Nazarov reaction belongs to the group of electrocyclic reactions and is one of the most versatile methods for the synthesis of five-membered rings, which are the key structural elements of numerous natural products. In general, the Nazarov cyclization can be catalyzed by Brønsted or Lewis acids. However, only a few asymmetric variations have been described, of which most require the use of large amounts of chiral metal complexes. The reactivities of Nazarov cyclizations are also depending on the substituents of the divinyl ketone substrates as described in the first chapter. The substrates to study Brønsted acid-catalyzed enantioselective Nazarov cyclization were prepared following the known procedures. The dihydropyran was treated with tBuLi in THF at –78 oC and then the α,β-unsaturated aldehydes 1 were added to the reaction mixture to afford the corresponding alcohols 2 in moderate to good yields. The alcohols 2 were oxidized to divinyl ketones 3 employing Dess-Martin periodinane/pyridine (DMP/py) in CH2Cl2 at room temperature to obtain the divinyl ketones 3 in moderate to good yields (Scheme 1). Scheme 1. Preparation of substrates in order to study Brønsted acid-catalyzed enantioselective Nazarov cyclization and subsequent transformations. At the starting point, an evaluation of suitable Brønsted acid catalysts for the enantioselective Nazarov cyclization of divinyl ketone 3a was performed. The initial reactions conducted with various BINOL-phosphoric acids 4a-4e in toluene at 60 oC provided the mixture of cis and trans cyclopentenones 5a with enantioselectivities of up to 82% ee (Table 1, entries 1-5). Eventually, improved reactivity could be achieved by using the corresponding N-triflylphosphoramides 4f and 4g, which even at 0 oC gave complete conversion after ten minutes. Additionally, it was shown that the use of these catalysts significantly enhanced both the diastereoselectivity (cis/trans ratio up to 7:1) and the enantioselectivity (up to 96% ee; Table 1, entries 6 and 7). Table 1. Evaluation of Brønsted acids 4a-4g in the enantioselective Nazarov cyclization. The scope of the Brønsted acid-catalyzed enantioselective Nazarov cyclization of various divinyl ketones 3 was explored under an optimized reaction condition (Scheme 2). Treatment of divinyl ketones 3 in CHCl3 in the presence of 2 mol% chiral BINOL-Ntriflylphosphoramide 4g at 0 oC for 1-6 h provided the corresponding cyclopentenone 5 in good yields (45-92%) with excellent enantioselectivities (up to 93% ee) (Scheme 2). Furthermore, the isomerization of cis-cyclopentenone under basic condition led to the corresponding trans-cyclopentenone without loss of enantiomeric purity. This efficient method introduced here was not only the first example of an organocatalytic electrocyclic reaction but also represented the first enantioselective activation of a carbonyl group catalyzed by a chiral BINOL phosphoric acid. Compared to the metal-catalyzed reaction, special features of this new Brønsted acid-catalyzed electrocyclization are the lower catalyst loadings (2 mol%), higher enantioselectivities, accessibility to all possible stereoisomers, as well as the mild conditions. ....
Large amplitude intramolecular motions in non-rigid molecules are a fundamental issue in chemistry and biology. The conventional approaches for study these motions by far-infrared and microwave spectroscopy are not applicable when the molecule is non-polar. Therefore, in the current thesis an alternative approach for the investigation of large amplitude intramolecular motions was developed and tested. This new method is based on femtosecond rotational degenerate four-wave mixing spectroscopy (fs DFWM), which is a particular implementation of rotational coherence spectroscopy. The method was successfully applied for the investigation of pseudorotation in pyrrolidine and the ring-puckering vibration in cyclopentene. Another important subject is the photophysics of molecules and molecular clusters which have an ultrashort lifetime of their electronically excited state (photoreactivity). These ultrashort lifetimes often represent a protective mechanism causing photostability. The photoreactivity is usually the manifestation either of an “elementary” reaction, such as proton or electron transfer, which occurs in the excited state or of a fast non-radiative deactivation processes, such as internal conversion via conical intersection of the electronically excited and ground state. Due to a short-lived excited state, the conventional vibrational spectroscopic methods, such as IR depletion detected by resonance two-photon ionization spectroscopy (IR/R2PI), are not applicable for the structural investigation of these systems. Therefore, new approach, termed IR depletion detected by multiphoton ionization with femtosecond laser pulses (IR/fsMPI), was developed for studying the structure of photoreactive microsolvated molecules. The IR/fsMPI technique was applied for investigating the clusters of 1H-pyrrolo[3,2-h]quinoline with water/methanol as well as adenine- and 9-methyl-adenine-hydrates. In addition, the excited state dynamics of bifunctional azaaromatic molecule 7-(2'-pyridyl)indole (7PyIn) was studied by femtosecond pump-probe resonance excitation multiphoton ionization technique (fs REMPI). Under electronic excitation of this molecule a fast proton transfer (phototautomerization) takes place, which is followed by radiationless excited state deactivation process. The fs REMPI spectra lead to the conclusion that the phototautomerization in 7PyIn is coupled with a twisting of the molecule, and that the twisting provides an efficient channel for ultrafast radiationless excited state deactivation. This pattern of excited-state tautomerization/deactivation might be quite general.
Epidermal growth factor (EGF) receptor belongs to the broad family of enzymatic receptors called receptor tyrosine kinases (RTKs). Generally, the binding of a ligand to these receptors leads to activation of their intracellular kinase activity that sets in motion a cascade of signaling events. In order to ensure appropriate responses to physiological stimuli, the cell is endowed with the ability to regulate signal transduction via numerous mechanisms such as dephosphorylation of the RTK and its substrates as well as downregulation of the RTK. Activation of EGFR is a potent mitogenic (proliferative) and motogenic (cell motility) signal that plays crucial roles during embryonic development and maintenance of adult tissue. EGFR signaling is primarily regulated by ligand-induced receptor internalization with subsequent degradation in lysosomes. While the complex of proteins that are recruited to EGFR after its activation is well understood, proteins that interact with the receptor in the absence of ligand binding are still not systematically studied. With the goal of identifying novel binding partners of non-activated EGFR, a membrane based yeast-two hybrid screen (MYTH) was conducted. MYTH is based on the principle of in vivo reconstitution of the N-terminus (Nub) and C-terminus (Cub) halves of ubiquitin once brought into close proximity. A chimeric protein consisting of EGFR fused to Cub and a transcription factor was used as a bait to screen Nub-tagged cDNA library. Analysis of resultant yeast transformants revealed a total of 87 proteins to interact with EGFR. Of these only 11 were previously shown to bind to EGFR. A majority of the other proteins were shown to interact with the receptor by yeast retransformation. Fifteen were confirmed to bind to EGFR by coimmunoprecipitation assays in mammalian cells. One of the novel EGFR interactors identified in the screen was histone deacetylase 6 (HDAC6). This deacetylase is localized in the cytoplasm and known to deacetylate alpha-tubulin, HSP90 and cortactin. The juxtamembrane region of EGFR binds to the Cterminus of HDAC6. Functionally, overexpression of wild type HDAC6 stabilized ligand-induced degradation of the receptor. On the other hand, deacetylase deficient or EGFR binding compromised mutants of HDAC6 were able to stabilize EGFR only partially. Downmodulation of HDAC6 expression by RNAi markedly accelerated degradation of the receptor. Taken together, HDAC6 is a negative regulator of EGFR downregulation that is dependent on its deacetylase activity and ability to bind to the receptor. Imaging studies revealed that HDAC6 does not affect internalization of EGFR from the plasma membrane but rather influences the post-endocytic trafficking of the receptor-ligand complex to lysosomes. Pulse-chase experiments using fluorophoretagged EGF showed that EGFR is transported faster towards the peri-nuclear region and delivered to late endosomes rapidly in HDAC6 depleted cells. HDAC6 is demonstrated to act, at least partly, by regulating the acetylation of alpha-tubulin. Upon EGFR activation, acetylation of alpha-tubulin on lysine 40 is progressively increased as shown by mass spectrometry and immunoblotting. Forced expression of a dominant negative mutant of alpha-tubulin, but not wild type alpha-tubulin, led to reduced speed and processive movement of early endosomes in GFP-Rab5 expressing cells. In a surprising twist, EGFR is able to phosphorylate HDAC6 on Tyr570. Phosphorylation of Tyr570 and Ser568 leads to inactivation of the deacetylase function of HDAC6 as shown by in vivo and in vitro assays. In summary, HDAC6 diminishes EGFR downregulation by slowing the transport of intracellular vesicles. The inhibitory effect is removed once HDAC6 is phosphorylated on key residues. In line with these findings, two recent reports have shown that hyper-acetylation of alpha-tubulin induced by inhibition of HDAC6 increases the transport of brain derived neurotrophic factor and JNK interacting protein-1 in different cell systems. Acetylated microtubules are more efficient in recruiting motor proteins like kinesin-1 and dynein. These findings indicate that HDAC6 plays an important regulatory role in intracellular trafficking pathways. However, several outstanding issues still remain unresolved. How does acetylation of microtubules influence vesicular trafficking? In this regard, the temporal and spatial dynamics of alpha-tubulin acetylation following EGFR activation should be studied. Furthermore, whether HDAC6 affects the trafficking of other endocytic cargos and additional organelles is an interesting question to address.
This thesis presents a 5.9 Å map of yeast FAS obtained by cryo-electron microscopy using single particle analysis (SPA). The EM-map has been analyzed both by quantitative and qualitative analysis to aid in understanding of the structure and dynamics of yeast FAS. This study approaches the factors limiting the resolution in EM (>20 Å) and further discusses the possibilities of achieving higher-resolutions (<10 Å) in cryo-EM by single particle analysis. Here, SPA is highlighted as a powerful tool for understanding the structure and dynamics of macro-molecular complexes at near native conditions. Though SPA has been used over the last four decades, the low-resolution range (20-30 Å) of the method has limited its use in structural biology. Over the last decade, sub nanometer resolution (<10 Å) structures solved by SPA have been reported --both in studies involving symmetric particles, such as GroEL (D7) and asymmetric particles, such as ribosomes (C1). Recently, near-atomic resolution in the range of 3.8-4.2 Å has been achieved in cases of highly symmetric icosahedral viral capsid structures as well. The yeast FAS structure (D3) presented here is one of two low symmetry structures submitted to the EM-database in a resolution range of 5-6 Å; the other being GroEL (D7). Fatty acid synthase (FAS) is the key enzyme for the biosynthesis of fatty acids in living organisms. There are two types of FAS, namely the type II FAS system in prokaryotes, consisting of a set of individual enzymes, and type I FAS found in eukaryotes as a multienzyme complex. Yeast fatty acid synthase (FAS) is a 2.6 MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a 3D map of yeast FAS at 5.9 Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fit well with the substratebinding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ~18 Å from the ACP substrate binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct insight into the proposed substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.