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The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
NMR and chromatography methods combined with mass spectrometry are the most important analytical techniques employed for plant metabolomics screening. Metabolomic analysis integrated to transcriptome screening add an important extra dimension to the information flow from DNA to RNA to protein. The most useful NMR experiment in metabolomics analysis is the proton spectra due the high receptivity of 1H and important structural information, through proton–proton scalar coupling. Routinely, databases have been used in identification of primary metabolites, however, there is currently no comparable data for identification of secondary metabolites, mainly, due to signal overlap in normal 1H NMR spectra and natural variation of plant. Related to spectra overlap, alternatively, better resolution can be find using 1H pure shift and 2D NMR pulse sequence in complex samples due to spreading the resonances in a second dimension. Thus, in data brief we provide a catalogue of metabolites and expression levels of genes identified in soy leaves and roots under flooding stress.
Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.
The research presented in this thesis characterizes U2AF homology motifs (UHM) and their interactions with UHM ligand motifs (ULM) in the context of splicing regulation. UHM domains are a subgroup of RNA recognition motifs (RRM) originally discovered in the proteins U2AF65 and U2AF35. Whereas canonical RRMs are usually involved in binding of RNA, UHM domains bind tryptophan containing linear protein motifs (ULM) instead. In the first article, we analyze the complex network of interactions between splicing factors and RNA that initiate the assembly of the spliceosome at the 3´ splice site of an intron. The protein U2AF65 binds a pyrimidine-rich element in introns and recruits U2snRNP by binding its protein component SF3b155. My contribution was to define the binding site of the protein U2AF65 to the intrinsically unstructured N-terminus of the scaffolding protein SF3b155. I could show that the UHM domain of U2AF65 recognizes a ULM in SF3b155, and that this binding site is not overlapping with the binding sites of other splicing factors, like p14, to SF3b155. As the U2AF65-UHM:SF3b155-ULM interaction is mutually exclusive with an interaction between U2AF65-UHM and a ULM in the splicing factor SF1, which was reported to initially recognize the branch point sequence, my results provide the molecular details on how SF3b155 replaces SF1 during spliceosomal reorganizations. In the second article, we show that overexpression of the UHM domain of the splicing factor SPF45 induces exon 6 skipping in the pre-mRNA of Fas (CD95/APO-1). I provide evidence for in vitro binding of SPF45-UHM to ULM sequences in the splicing factors U2AF65, SF1, and SF3b155. I crystallized free and SF3b155-bound SPF45 UHM and solved both structures by X-ray crystallography. The analysis of the complex interface and sequence differences in the ULMs allowed me to design mutations of SPF45-UHM, which selectively inhibit binding to distinct ULMs. After assessing the ULM binding properties in vitro, we could show that the activity of SPF45-UHM in influencing the splicing pattern of Fas relies on interactions with SF3b155 and/or SF1, but that an interaction with U2AF65 is dispensable. A mechanism for the activity of SPF45-UHM could thus be engaging in ULM interactions and thus interfering with the network of interactions that initiate the assembly of the spliceosome at the 3´splice site, as described above. In the third article, we describe an unusual flexible homodimerization mode of the UHM in the splicing factor Puf60, which enables simultaneous interactions with ULM sequences on other splicing factors. I could show that the NMR relaxation properties of Puf60-UHM are inconsistent with a model of a rigid dimer, but rather indicate a dimerization via a flexible linker. I identified a flexible loop in the peptide backbone of Puf60-UHM, and showed that mutiation of acidic residues in this loop impairs the dimerization. To analyze the dimerization interface in further detail, I solved the structure of Puf60-UHM by X-ray crystallography. The acidic residues in the flexible loop of one UHM dimer subunit mediate the dimerization by contacting basic residues on the β-sheet surface of the other dimer subunit. Differences in the four dimer interfaces observed for the eight molecules in the asymmetric unit of the crystal support the model of an undescribed, flexible mode of dimerization, and thus complement the NMR relaxation data. Furthermore, I could show that the Puf60-UHM dimer and U2AF65-UHM contact different ULM sequences on the SF3b155 N-terminus in vitro, thus providing a possible explanation for the mutual cooperative activation of Puf60 and U2AF65 in splicing assays described in the literature. The fourth article is a review about recent research on the recognition of DNA double strand breaks (DSB) by covalent histone modifications. The p53 binding protein 1 (53BP1) is a DSB sensor and a checkpoint protein for mitosis. Recent crystallographic evidence indicates that 53BP1 recognizes DSB sites by binding histone H4 dimetylated at lysine 20 (H4-K20). We provide a comprehensive overview of the atomic resolution structures that revealed how proteins can specifically recognize histone tail modifications, especially methylated lysines, to read the information stored in what is called the histone code.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
Excitation of CO molecules into the lowest vibrational level of the B1Σ+ electronic state by absorption of the (B 1Σ+υ′=0 →X 1Σ+ ,υ′′=0) resonance band at 1150 Å has been studied under various experimental conditions by observing the steady state fluorescence of the (B 1Σ+→A1Π) Angstrom bands. Stern-Volmer plots of the fluorescence intensities at the addition of various foreign gases yielded straight lines whose slopes k̃qм = kqм · τeff were strongly dependent on the CO sample pressure. This effect was found to be due to changes of the effective radiative lifetime of the B 1Σ+υ′=0 because of resonance trapping of the (0,0) band of the (B → X) fluorescence. The CO(B 1Σ+υ′=0) molecules are found to be quenched by He, Ne, Ar, H2 and D2 with effective collision cross sections of 0.23, 0.48, 22.4, 10.7, and 11.4 Å2, respectively, at 298 °K. In addition, an approximate value for the ratio ABA/ (ABA+ABX)of the radiative transition probabilities of the (B → A) and (B → X) transitions could be derived from the measurements.
Das OH-Radikal, bestehend aus einem Sauerstoff- und einem Wasserstoffatom, ist verantwortlich für den Selbstreinigungsmechanismus der Atmosphäre. Als Oxidationsmittel reagiert es mit praktisch allen Spurengasen, wie z. B. dem giftigen Kohlenmonoxid, dem Treibhausgas Methan und dem Schwefeldioxid, und macht sie wasserlöslich, so daß sie im Regen gelöst ausgewaschen werden können (Waschmitteleffekt). Welche Schlüsselstellung das OH-Radikal in der Atmosphärenchemie hat, beschreibt der Chemiker Franz Josef Comes. Gleichzeitig stellt er das weltweit empfindlichste Absolutverfahren zur Bestimmung von troposphärischen OH-Konzentrationen vor, das in Frankfurt entwikkelt und zur Zeit getestet wird.
Photofragmentation spectroscopy—the study of "half collisions" with polarized light of subdoppler line width—opens a window to look into the structure of molecules. The energy partitioning among the particular degrees of freedom of the products of the fragmentation reaction is described by the scalar properties, the direction and magnitude of a particular type of motion is described by the vector properties. The measurement of the scalar and vector properties allows a pictorial view of the intermediate state. The forces which make the fragments fly apart or rotate and vibrate can be "seen" from the line shapes. Information on the unstable intermediate state is gained from the stable fragments long after the dissociation of the parent molecule. In particular, information on the "lifetime" of the intermediate on a femtosecond time scale can be obtained.
A number of molecules, mainly three and four atomic, have been studied by this technique. Hydrogen peroxide has shown up as a textbook example. A complete analysis was possible including not only correlation of different types of fragment motion but also a correlation of the two coincident particles formed from the same parent molecule. The experimental results are in full agreement with recent calculations of the dynamics of the fragmentation on newly obtained potential energy surfaces. Hydrogen peroxide shows a strong dependence of its potential energy on the dihedral angle in the two electronic states amenable to laser excitation. This experiment further demonstrates that an analysis is also possible if two states are excited simultaneously.
Another good example is the fragmentation of hydrazoic acid for which also coincident pair correlation has been treated. Here again the results agree excellently with a qualitative picture which can be drawn from recently calculated ab initio potential energy surfaces. The HN3 example is much more complicated than the former one due to its higher structured upper potential energy surface. Strong rotational excitation is observed in the N2 fragment leaving the NH fragment rotationally cold.
The treatment of vector correlations in molecular photofragmentation is a powerful tool for the study of the dynamics of molecular dissociation reactions.