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81.
82.
NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings 总被引:1,自引:0,他引:1
A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar 1H-1H coupling constants with respect to the sign of one-bond 1H-X coupling constants in linear three-spin systems X-1H-1H, where X = 13C or 15N. In contrast to the original JHH-TOCSY experiments, the signs of J
HH couplings may be determined for CH2-CH2 moieties and for uniformly 13C/15N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum. 相似文献
83.
Cherepanov DA Bibikov SI Bibikova MV Bloch DA Drachev LA Gopta OA Oesterhelt D Semenov AY Mulkidjanian AY 《Biochimica et biophysica acta》2000,1459(1):10-34
After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle. 相似文献
84.
Patrik Andersson Kerstin Nordstrand Maria Sunnerhagen Edvards Liepinsh Ivars Turovskis Gottfried Otting 《Journal of biomolecular NMR》1998,11(4):445-450
Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,1 H-detected 15N-1H or13 C-1H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra. 相似文献
85.
86.
Patrik Andersson Bernard Gsell Beat Wipf Hans Senn Gottfried Otting 《Journal of biomolecular NMR》1998,11(3):279-288
An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N, 1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D15 N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B. 相似文献
87.
Markus Düchler Fritz Schmoll Franz Pfneisl Gottfried Brem Karl Schellander 《Biology of the cell / under the auspices of the European Cell Biology Organization》1998,90(2):199-205
We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow rapidly on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO-1, a tight-junction associated protein, but negative immunostaining for an anti-vimentin antibody. In confluent cell monolayers ‘domes’ became visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end-buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of β-lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line. 相似文献
88.
89.
Katrin Spiesberger Florian Paulfranz Anton Egger Judith Reiser Claus Vogl Judith Rudolf-Scholik Corina Mayrhofer Ludger Grosse-Hovest Gottfried Brem 《PloS one》2015,10(10)
Background
30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).Results
With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Conclusion
Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. 相似文献90.
Klaudia Schossleitner Andreas Habertheuer Richard Finsterwalder Heinz P. Friedl Sabine Rauscher Marion Gr?ger Alfred Kocher Christine Wagner Stephan N. Wagner Gottfried Fischer Marcus J. Schultz Dominik Wiedemann Peter Petzelbauer 《PloS one》2015,10(11)