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Metal-organic frameworks (MOFs) have emerged as a promising class of crystalline porous inorganic-organic hybrid materials showing a wide range of applications. In order to realize the integration of MOFs into specific devices, this thesis mainly focuses on the controlled growth and the properties of highly oriented surface-mounted metal-organic frameworks (SURMOFs).
The stepwise layer-by-layer (LbL) growth method exhibits vast advantages for the controllable growth of SURMOFs regarding the crystallite orientation, film thickness and homogeneity. However, up to date, only a few MOFs have been demonstrated to be suited for this protocol. So the first project of this thesis was designed to extend the applicability of the LbL growth. To this end, a semi-rigid linker based [Cu2(sdb)2(bipy)] (sdb = 4,4’-sulfonylbiphenyl dicarboxylate; bipy = 4,4’-bipyridine) MOF was chosen. Employing the LbL growth, [Cu2(sdb)2(bipy)] SURMOFs were successfully grown onto both pyridyl- and carboxyl-terminated surfaces at the temperature range of 15-65 °C. Interestingly, the orientation of the SURMOFs largely depends on temperature on both surfaces. At low temperatures (below 40 °C), SURMOFs with exclusive [010] orientation are obtained. In contrast, at high temperatures (40-65 °C), [001] oriented SURMOF growth is favored. A novel growth mode was demonstrated, which is, instead of surface chemistry, the temperature-induced ripening processes and the tendency to minimize surface energies can dominate the SURMOF growth.
Inspired by the advantages of LbL deposition of isoreticular SURMOFs, the second project was conceived to grow multivariate SURMOFs (MTV-SURMOFs) using mixed dicarboxylate linkers. We advance a hypothesis that linker acidity (expressed by the pKa values) may have an influence on the oriented growth of MTV-SURMOFs. To test the hypothesis, seven isoreticular [Cu2L2(dabco)] (L = single kind of dicarboxylate linker; dabco = 1,4-diazabicyclo[2.2.2]octane) SURMOFs were grown onto pyridyl-terminated surfaces at 60 °C. The quality of [001] orientation is greatly affected by the acidity of the linkers. With this observation, we deposited a series of [Cu2Lm2(dabco)] (Lm = mixed dicarboxylate linkers) SURMOFs under the same conditions. [Cu2Lm2(dabco)] SURMOFs with exclusive [001] orientation are obtained when the growth solution contains two linkers of relatively high pKa value or more than two kinds of linkers (independent of the pKa values), while the mixtures of ligands with relatively low pKa values or a high content of low pKa valued linkers can result in mis-oriented growth of SURMOFs with unexpected [100] orientation.
Moreover, the LbL growth shows enormous potential in the rational construction of functional SURMOFs. Therefore, the third project of this thesis was devised to deposit SURMOFs containing redox-active species. For this, the 4,4’-biphenyldicarboxylic acid (H2(bpdc)) linker was functionalized with ferrocene (Fc) and dimethyl ferrocene (Me2Fc) moieties. [Cu2(bpdc-amide-Fc)2(dabco)] SURMOF (Fc-SURMOF) is perfectly grown along the [100] direction, while mis-oriented growth of [Cu2(bpdc-amide-Me2Fc)2(dabco)] SURMOF (Me2Fc-SURMOF) was observed. Surprisingly, Fc-SURMOF shows excellent electrochemical properties due to the reversible oxidation and reduction of the ferrocene moieties in the oriented pores, while the Me2Fc-SURMOF was found to be a closely packed insulating layer since no extensive charge transfer is observed. A diffusion controlled mechanism of redox reaction is proposed, where the diffusion of the counter anions in the pores limits the current.
Besides the LbL growth protocol, the spin-coating technique is also promising for the oriented growth of SURMOFs. Driven by the specific applications, the fourth project of this thesis was planned to grow functional SURMOFs containing catalytically active units. The Keggin-type polyoxometalates (POMs) with high catalytic activities were chosen to functionalize the HKUST-1 SURMOFs. Combining the technique with methanol vapor induced growth, a series of POM functionalized HKUST-1 SURMOFs (denoted as POM@HKUST-1 SURMOFs) were controllably deposited onto pyridyl-terminated surfaces. The SURMOFs exhibit great potential as electrocatalysts in electrochemical devices due to the excellent redox properties of POMs. In addition, the PTA@HKUST-1 (PTA = phosphotungstic acid) SURMOF can be employed as an ideal platform for the selective loading of methylene blue (MB) dye with high efficiency. Owing to the strong binding between the dye molecules and the framework, the MB dye cannot be desorbed by ion exchange and MB loaded PTA@HKUST-1 SURMOF shows reliable redox properties under inert conditions, further confirming the application potential in electrochemical devices.
Background: How a dentist works, such as the patterns of movements performed daily, is also largely affected by the workstation Dental tasks are often executed in awkward body positions, thereby causing a very high degree of strain on the corresponding muscles. The objective of this study is to detect those dental tasks, during which awkward postures occur most frequently. The isolated analysis of static postures will examine the duration for which these postures are maintained during the corresponding dental, respectively non-dental, activities.
Methods: 21 (11f/10 m) dentists (age: 40.1 ± 10.4 years) participated in this study. An average dental workday was collected for every subject. To collect kinematic data of all activities, the CUELA system was used. Parallel to the kinematic examination, a detailed computer-based task analysis was conducted. Afterwards, both data sets were synchronized based on the chronological order of the postures assumed in the trunk and the head region. All tasks performed were assigned to the categories "treatment" (I), "office" (II) and "other activities" (III). The angle values of each body region (evaluation parameter) were examined and assessed corresponding to ergonomic standards. Moreover, this study placed a particular focus on static positions, which are held statically for 4 s and longer.
Results: For "treatment" (I), the entire head and trunk area is anteriorly tilted while the back is twisted to the right, in (II) and (III) the back is anteriorly tilted and twisted to the right (non-neutral position). Static positions in (I) last for 4–10s, static postures (approx. 60%) can be observed while in (II) and (III) in the back area static positions for more than 30 s are most common. Moreover, in (II) the back is twisted to the right for more than 60 s in 26.8%.
Conclusion: Awkward positions are a major part of a dentists’ work. This mainly pertains to static positions of the trunk and head in contrast to "office work." These insights facilitate the quantitative description of the dentist profession with regard to the related physical load along with the health hazards to the musculoskeletal system. Moreover, the results allow for a selective extraction of the most unfavorable static body positions that dentists assume for each of the activities performed.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I1III2 assembly in bovine heart and the yeast Yarrowia lipolytica, but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis. Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III2, but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer.
The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.
Für die Optimierung sowie Entwicklung lichtsteuerbarer Systeme für biologische Anwendungen oder neue Materialien ist ein detailliertes Verständnis der zugrunde liegenden komplexen, lichtinduzierten Prozesse eine Voraussetzung. Die Verwendung von Photoschaltern in Makromolekülen ermöglicht eine zeitliche und örtliche Kontrolle über strukturelle Änderungen sowie die entsprechend folgenden (biologischen) Funktionen durch die Verwendung von Licht als externem Auslöser.
Ein wichtiger Bestandteil dieser Arbeit befasst sich mit der Entwicklung eines auf Licht reagierenden Riboschalters, welcher die gezielte Kontrolle über Genexpression ermöglicht. Hierzu wurde eine spektroskopische Charakterisierung von verschiedenen Photoschaltern bezüglich einer Verwendung als biologischer Ligand sowie der Wechselwirkungen zwischen Azobenzolen und RNA, auch hinsichtlich ihrer Bindungsdynamiken durchgeführt. Zunächst wurde die hohe Abhängigkeit der (photo-)chemischen Eigenschaften der Azobenzole von der Wahl der Substituenten untersucht, wobei besonders die Anwendung in wässrigem Milieu betrachtet wurde. In einer detaillierten (zeitaufgelösten) Studie wurde der positionsabhängige Einfluss der Hydroxy-Substitution von Azobenzolen auf die Photoisomerisierung in wässriger Lösung untersucht. Für eine ortho-Substitution ergab sich hierbei ein alternativer Deaktivierungskanal nach Photoanregung, welcher stärker ausgeprägt ist als die Isomerisierung. Hierbei wird ein intramolekularer Protontransfer im angeregten Zustand (ESIPT) beobachtet, welcher mit einer Zeitkonstante von 0.3 ps beschrieben werden kann und in einer Keto-Spezies resultiert. Eine Keto-Enol-Tautomerie konnte für die para-Hydroxy-Substitution schon im Grundzustand beobachtet werden. Somit können beide Spezies gezielt adressiert werden. Durch Acetylierung der Hydroxygruppe verlangsamt sich die thermische Relaxation des cis-Isomer zu dem entsprechenden trans-Isomer signifikant ohne die Isomerisierung zu beeinträchtigen. Dementsprechend ermöglicht eine solche Acetylierung die Verwendung von bekannten Azobenzolderivaten als Photoschalter.
Zudem werden in dieser Arbeit zwei verschiedene Herangehensweisen in der Entwicklung eines Riboschalters beschrieben, welcher sich durch Licht regulieren lässt.
Diese sind durch kovalentes bzw. nicht-kovalentes Einbringen eines Azobenzolderivats in die RNA Struktur charakterisiert. Ein neuer Linker, welcher auf einer Desoxyribose-Struktur beruht, wird für die kovalente Anbindung des Azobenzols an den RNA Strang präsentiert, welcher eine licht-induzierte Dehybridisierung ermöglichen soll. Eine außergewöhnlich hohe Schaltamplitude mit einem cis-Gehalt von etwa 90% konnte für das Azobenzol im RNA Einzelstrang schon bei Raumtemperatur ermittelt werden. Zudem wurde der Einfluss des Photoschalters sowie der benachbarten Nukleotide in der RNA auf die Stabilität der RNA Doppelhelix untersucht. Die zweite Vorgehensweise beruht auf einer nicht-kovalenten Bindung zwischen einem Azobenzolderivat und einem RNA-Aptamer, welche lediglich für eines der Photoisomere ermöglicht wird, wodurch eine örtliche und zeitliche Kontrolle der Ligandenbindung der RNA erfolgt. Im Rahmen dieser Arbeit war es möglich zwei verschiedene photoschaltbare RNA Aptamere zu identifizieren und zu untersuchen, welche eine hohe Spezifität und Affinität aufweisen. Zudem wurde die Photoisomerisierung des Azobenzols innerhalb der RNA-Struktur sowie daraus resultierende lichtinduzierte Konformationsänderungen der RNA mittels zeitaufgelöster Anreg-/Abtastspektroskopie untersucht. Die daraus resultierende Dynamik der photoinduzierten Ligandenbindung sollte eine weitere gezielte Optimierung lichtschaltbarer biologischer Systeme erlauben.
Der zweite Teil dieser Arbeit beschäftigt sich mit der zeitaufgelösten Untersuchung eines photoschaltbaren Foldamers. Speziell wurde der strukturelle Übergang des OmPE-Foldamers 10-5 zwischen einer definierten helikalen und einer ungefalteten Konformation auf Grund der Photoisomerisierung der, in das Rückgrat integrierten, Azobenzole untersucht.
Dabei konnten die frühen (Ent-)Faltungsmechanismen des Foldamers im sub-Nanosekunden-Zeitbereich beobachtet werden, welche durch quantenmechanische Rechnungen unterstützt werden konnten. Darüberhinaus, war es möglich einen Anregungsenergietransfer vom PE-Rückgrat des Foldamers auf die Azobenzole nachzuweisen, welcher die Lebensdauer der angeregten Zustände des Systems signifikant verkürzt.
Diese Arbeit liefert wichtige Informationen zu den Reaktionspfaden, den gezielten Wechselwirkungen zwischen Photoschaltern und größeren organischen Molekülen, sowie den daraus resultierenden lichtinduzierten strukturellen Änderungen durch die Anwendung einer Vielzahl an (zeitaufgelösten) spektroskopischen Methoden. Diese Ergebnisse tragen zum weiteren Verständnis komplexer Prozesse in biologischem sowie nicht-biologischem Zusammenhang und somit zu einer weiterführenden Entwicklung neuer Systeme bei.
The layer‐by‐layer (LbL) method is a well‐established method for the growth of surface‐attached metal–organic frameworks (SURMOFs). Various experimental parameters, such as surface functionalization or temperature, have been identified as essential in the past. In this study, inspired by these recent insights regarding the LbL SURMOF growth mechanism, the impact of reactant solutions concentration on LbL growth of the Cu2(F4bdc)2(dabco) SURMOF (F4bdc2−=tetrafluorobenzene‐1,4‐dicarboxylate and dabco=1,4‐diazabicyclo‐[2.2.2]octane) in situ by using quartz‐crystal microbalance and ex situ with a combination of spectroscopic, diffraction and microscopy techniques was investigated. It was found that number, size, and morphology of MOF crystallites are strongly influenced by the reagent concentration. By adjusting the interplay of nucleation and growth, we were able to produce densely packed, yet thin films, which are highly desired for a variety of SURMOF applications.
A small single molecule with multiple photoswitchable subunits, selectively and independently controllable by light of different wavelengths, is highly attractive for applications in multi-responsive materials and biological sciences. Herein, triple photoswitches are presented consisting of three independent azobenzene (AB) subunits that share a common central phenyl ring: the meta-trisazobenzenes (MTA). It is the unique meta-connectivity pattern leading to decoupling of all azo-subunits although they do overlap spatially. Based on this pattern, we design a triple MTA photoswitch, as proof-of-principle, with three different, electronically independent AB branches on the computer, which can be individually photo-excited to trigger ultra-fast E → Z isomerization at the selected AB branch.
The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.
Highlights
• Cryo-EM structures of the yeast low-affinity phosphate importer ScPho90
• Complementary structures reveal insights into the substrate translocation mechanism
• Comparisons with homologous transporters highlight the conserved transport mechanism
• Regulation by the SPX domain is discussed
Summary
Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters.
We compiled an NMR data set consisting of exact nuclear Overhauser enhancement (eNOE) distance limits, residual dipolar couplings (RDCs) and scalar (J) couplings for GB3, which forms one of the largest and most diverse data set for structural characterization of a protein to date. All data have small experimental errors, which are carefully estimated. We use the data in the research article Vogeli et al., 2015, Complementarity and congruence between exact NOEs and traditional NMR probes for spatial decoding of protein dynamics, J. Struct. Biol., 191, 3, 306–317, doi:10.1016/j.jsb.2015.07.008 [1] for cross-validation in multiple-state structural ensemble calculation. We advocate this set to be an ideal test case for molecular dynamics simulations and structure calculations.
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
The four subunit (SU) aa3 cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. It uses electrons from cytochrome c to reduce molecular oxygen to water. Its binuclear active center, residing in SU I, contains hemeÊa3 and CuB, the latter being liganded by three histidine residues. Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity.
To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT CcO was constructed, carrying the gene for SUÊI on a low copy number plasmid. This rec. WT showed, as expected, no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential overproduction of SUÊI due to plasmid coding and the resulting deficiency in metal inserting chaperones might impair the correct insertion of hemeÊa3 and CuB because of a deficiency in metal inserting chaperones. This in turn might lead to differences in side chain orientation and to changes in the water network. However, slight changes might cause an increased accessibility of the active center for hydrogen peroxide, resulting in an increased catalase activity. The availability of chaperones and therefore the proposed structural reasons for the difference was improved by cloning the genes for the two metal inserting chaperones CtaG and Surf1c on the same plasmid together with SUÊI. This new rec. WT CcO showed in fact a reduced catalase activity. Another WT with a deletion in the chromosomal second, non expressing gene of SU I was analysed to prove plasmid coding as the reason for the difference of the ATCC WT and the rec. WT. This strain showed an increased kcat of the catalase activity as well, additionally pointing to a regulatory effect of the non expressed gene for SU I in the chromosome. To fathom the structural difference of the increased catalase activity, differential scanning calorimetry was used, but no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging, the thermal stability of the rec. WT CcO declined faster than that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.
To characterize the catalase reaction, several known inhibitors were used to probe the contribution of the different metal cofactors in the catalase reaction. In addition variants in aromatic amino acids near the active center were constructed to conclude on a possible reaction mechanism of the catalase activity of CcO. These variants in combination with the wild type forms were analysed for radical signals by EPR-spectroscopy. A radical relevant for the catalase reaction of CcO was found in the F-intermediate of all variants and all wild type forms. This narrow 12 G radical signal was assigned to a porphyrine radical probably involved in the catalase reaction of CcO. Moreover, gas chromatography-mass spectrometry measurements were used to analyse isotopically labelled oxygen produced in the catalase reaction.
As a result of these experiments, a reaction cycle of the catalase activity of CcO is postulated and the structural difference between the ATCC and rec. WT CcO is outlined. The catalase activity appears to be a true catalase activity and not a "pseudocatalase" activity.
Membrane proteins are biological macromolecules that are located in a cell’s membrane and are responsible for essential functions within an organism, which makes them to prominent drug targets. The extraction of membrane proteins from the hydrophobic membrane bilayer to determine high-resolution crystal structures is a difficult task and only 2% of all solved proteins structures are membrane proteins. Computational methods may help to gain deeper insights into membrane protein structures and their functions. This study will give an overview of such computational methods on a representative set of membrane proteins and will provide ideas for future computational and experimental research on membrane proteins.
In a first step (chapter 2), I updated an earlier, manually-curated data set of homologous membrane proteins (HOMEP) to more recent versions in 2010 (HOMEP2) and 2013 (HOMEP3) using an automated clustering approach. High-resolution structures of membrane proteins listed in the PDB_TM database were structurally aligned and subsequently clustered using structural similarity scores. Both data sets were used as a standard gold reference set for subsequent work.
Subsequently, I have updated and applied the sequence alignment program AlignMe to determine protein descriptors that are suitable for detecting evolutionary relationship between homologous a-helical membrane proteins. Single input descriptors were tested alone and in combination with each other in different modes of AlignMe by optimizing gap penalties on the HOMEP2 data set. Most accurate alignments and homology models on the HOMEP2 data set were observed when using position-specific substitution information (P), secondary structure propensities (S) and transmembrane propensities (T) in the AlignMe PST mode. An evaluation on an independent reference set of membrane protein sequence alignments from the BAliBASE collection showed that different modes of AlignMe are suitable for different sequence similarity levels. The AlignMe PST mode improved the alignment accuracy significantly for distantly related proteins, whereas for closely-related proteins from the BAliBASE set the AlignMe PS mode was more suitable. This work was published in March 2013 in PLOS ONE. In order to allow also an easier usage of the AlignMe program, I have implemented a web server of AlignMe (chapter 4) that provides the optimized settings and gap penalties for the AlignMe P, PS and PST modes. A comparison to other recent alignment web server shows that the alignments of AlignMe are similar or even more accurate than those of other methods, especially for very distantly related proteins for which the inclusion of membrane protein information has been shown to be suitable. This work was published in the NAR web server issue in July 2014.
Although membrane-specific information has been shown to be suitable for aligning distantly related membrane proteins on a sequence level, such information was not incorporated into structural alignment programs making it unclear which method is the most suitable for aligning membrane proteins. Thus, I compared 13 widely-used pairwise structural alignment methods on an updated reference set of homologous membrane protein structures (HOMEP3) and evaluated their accuracy by building models based on the underlying sequence alignments and used scoring functions (e.g., AL4 or CAD-score) to rate the model accuracy (chapter 5). The analysis showed that fragment-based approaches such as FR-TM-align are the most useful for aligning structures of membrane proteins that have undergone large conformational changes whereas rigid approaches were more suitable for proteins that were solved in the same or a similar state. However, no method showed a significant higher accuracy than any other. Additionally, all methods lack a measure to rate the reliability of the accuracy for a specific position within a structure alignment. In order to solve these problems, I propose a consensus-type approach that combines alignments from four different methods, namely FR-TM-align, DaliLite, MATT and FATCAT and assigns a confidence value to each position of the alignment that describes the agreement between the methods. This work has been published 2015 in the journal “PROTEINS: structure, function and bioinformatics”.
Consensus alignments were then generated for each pair of proteins of the HOMEP3 data set and subsequently analyzed for single evolutionary events within membrane spanning segments and for irregular structures (e.g., 310- and p-helices) (chapter 6). Interestingly, single insertions and deletions could be observed with the help of consensus alignments in the conserved membrane-spanning segments of membrane proteins in four protein families. The detection of such single InDels might help to identify crucial residues for a proteins function.
For a certain class of ocean models describing the exchange of inorganic carbon between the atmosphere and the surface layer of the ocean as well as between the surface layer and the deep sea the dynamical airborne fraction is evaluated analytically under the assumption that the growth rate of the atmospheric source term (fossil fuel plus net biogenic carbon input into the atmosphere) is slowly variable with time. Each of these models exhibits a certain uptake capacity of the deep ocean which is quantified. Considerations are made as to whether the terrestrial biota are to be regarded as a source or a sink for additional atmospheric CO2 depending on the modelling of the deep ocean. It is shown that a global one-dimensional box-diffusion ocean model with a depth dependent eddy diffusivity K(z) - K(0) exp[-z/z*], with an adjustable parameter set {K(0), z*}, provides a fairly well fit to the prebomb 14C ocean distribution and to an appreciable net biogenic carbon transfer into the atmosphere. The range of future atmospheric CO2 partial pressures is estimated for a given fossil input.
The adaptive response of Sorghum bicolor landraces from Egypt to drought stress and following recovery was analyzed using two-dimensional difference gel electrophoresis, 2D-DIGE. Physiological measurements and proteome alterations of accession number 11434, drought tolerant, and accession number 11431, drought sensitive, were compared to their relative control values after drought stress and following recovery. Differentially expressed proteins were analysed by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS. Alterations in protein contents related to the energy balance, metabolism (sensu Mewes et al. 1997), and chaperons were the most apparent features to elucidate the differences between the drought tolerant and sensitive accessions. Further alterations in the levels of proteins related to transcription and protein synthesis are discussed.
pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.
N-Allyltetramethylpiperidine is readily isomerized to the corresponding enamine by treatment with catalytic amounts of B(C6F5)3. It adds HB(C6F5)2 at the nucleophilic enamine carbon atom to form a C/B Lewis adduct. This reacts with two molar equivalents of carbon monoxide by selective head to tail coupling to give a five-membered C2O2B heterocycle. In contrast the enamine/HB(C6F5)2 Lewis pair reacts with two molar equiv. of nitric oxide by head to head coupling. This reaction probably proceeds via equilibrium with the corresponding vicinal N/B Lewis pair. Most products were characterized by X-ray diffraction.
The title co-crystal, 1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane (HMTA, 1)–4-fluorophenol (4-FP) (1/1), C6H12N4·C6H5FO, shows an unusual asymmetric unit that comprises eight independent molecules (Z′′ = 8), four for each component, with four formula units per asymmetric unit (Z′ = 4). In the molecular packing, each HMTA molecule bridges one 4-FP molecule via an O−H···N hydrogen bond to form a two-molecule aggregate. Differences can be observed between the bond lengths and angles of the independent HMTA and 4-FP molecules and those of the molecules in the aggregate. The C−N bonds exhibit different bond lengths in the tetrahedral cage-like structure of the HMTA molecules, but the largest differences between the molecular aggregates are in the bond lengths in the 4-fluorophenol ring. In the crystal, the HMTA and 4-FP molecules form two hydrogen-bonded (O−H···N, C−H···F and C−H···O) dimers of HMTA and 4-FP molecules, A···D and B···C inversion dimers, which generate enlarged R88(34) ring motifs in both supramolecular structures. In both structures, the crystal packing also features additional C−H···F and C−H···O interactions. The A···D and B···C dimers are linked by additional C−H···F and C−H···O hydrogen bonds, forming columns along the a and b axes, respectively. The importance of the C−H···F interaction to the structure and crystal packing has been demonstrated.
We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >>>CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum.