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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.