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41.
Franz Tumulka Christian Roos Frank Löhr Christoph Bock Frank Bernhard Volker Dötsch Rupert Abele 《Journal of biomolecular NMR》2013,57(2):141-154
The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence. 相似文献
42.
43.
Herget M Oancea G Schrodt S Karas M Tampé R Abele R 《The Journal of biological chemistry》2007,282(6):3871-3880
By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family. 相似文献
44.
Goncalo L. Silva Jacek Plewka Helga Lichtenegger Ana C. Dias-Cabral Alois Jungbauer Rupert Tscheließnig 《Biotechnology and bioengineering》2019,116(1):76-86
Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects. 相似文献
45.
Sophie Winkler Rupert Derler Bernd Gesslbauer Elmar Krieger Andreas J. Kungl 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(3):528-533
Background
Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues.Methods
A disaccharide compositional analysis of the HS dp6 fraction in combination with MS analysis of the CCL2-depleted dp6 fraction was the basis for target GAG ligand structure suggestions. Four experimentally-derived heparan sulfate hexasaccharides, two potentially chemokine-specific and two unspecific, have been docked to CCL2. Subsequent 300?ns molecular dynamics simulations were used to improve the docked complexes.Results
Hexasaccharides with four sulfations and no acetylations are suggested for selective and high affinity chemokine binding. Using the Antithromin-III/heparin complex as positive control for docking, we were able to recover the correct complex structure only if the previously liganded ATIII structure was used as input. Since the liganded structure is not known for a CCL2-GAG complex, we investigated if molecular dynamics simulations could improve initial docking results. We found that all four GAG oligosaccharides ended up in close contact with the known binding residues after about 100?ns simulation time.Conclusions
A discrimination of specific vs. unspecific CCL2 GAG ligands is not possible by this approach. Long-time molecular dynamics simulations are, however, well suited to capture the delicate enthalpy/entropy balance of GAG binding and improve results obtained from docking.General significance
With the comparison of two methods, MS-based ligand identification and molecular modelling, we have shown the current limitations of our molecular understanding of complex ligand binding which is could be due to the numerical inaccessibility of ligand-induced protein conformational changes. 相似文献46.
47.
Hochachka PW Rupert JL Monge C 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》1999,124(1):1-17
Analysis of human responses to hypobaric hypoxia in different lineages (lowlanders, Andean natives, Himalayan natives, and East Africans) indicates 'conservative' and 'adaptable' physiological characters involved in human responses to hypoxia. Conservative characters, derived by common descent, dominate and indeed define human physiology, but in five hypoxia response systems analyzed, we also found evidence for 'adaptable' characters at all levels of organization in all three high altitude lineages. Since Andeans and Himalayans have not shared common ancestry with East Africans for most of our species history, we suggest that their similar hypoxia physiology may represent the 'ancestral' condition for humans--an interpretation consistent with recent evidence indicating that our species evolved under 'colder, drier, and higher' conditions in East Africa where the phenotype would be simultaneously advantageous for endurance performance and for high altitude hypoxia. It is presumed that the phenotype was retained in low capacity form in highlanders and in higher capacity form in most lowland lineages (where it would be recognized by most physiologists as an endurance performance phenotype). Interestingly, it is easier for modern molecular evolution theory to account for the origin of 'adaptable' characters through positive selection than for conserved traits. Many conserved physiological systems are composed of so many gene products that it seems difficult to account for their unchanging state (for unchanging structure and function of hundreds of proteins linked in sequence to form the physiological system) by simple models of stabilizing selection. 相似文献
48.
Knittel T Kobold D Piscaglia F Saile B Neubauer K Mehde M Timpl R Ramadori G 《Histochemistry and cell biology》1999,112(5):387-401
Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded
as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features
defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically
carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC
[desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth
muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of
central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase
in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected
within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar–parenchymal interface, while
rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC,
were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct
compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in
the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation.
Accepted: 16 September 1999 相似文献
49.
Constitutional Mutations of the hSNF5/INI1 Gene Predispose to a Variety of Cancers 总被引:8,自引:0,他引:8 下载免费PDF全文
Nicolas Svenet Eammon Sheridan Daniel Amram Pascale Schneider Rupert Handgretinger Olivier Delattre 《American journal of human genetics》1999,65(5):1342-1348
Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations. 相似文献
50.
Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development. We describe a device to monitor these oscillations in several samples in parallel. The apparatus consists of a thermostated cuvette holder where up to eight cuvettes containing cell suspension are inserted. Cells are aerated and kept in suspension via an airlift. Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes. The resulting signal is digitized and recorded with a sampling rate of two measuring points/second. The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point. This represents an advantage over the conventional single cuvette approach, as oscillation characteristics themselves are developmentally regulated. Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples, the behavior of wild-type and several mutant strains can be compared under identical experimental conditions. 相似文献