000837200 001__ 837200 000837200 005__ 20210129231224.0 000837200 0247_ $$2ISSN$$a1866-1785 000837200 020__ $$a978-3-89336-683-5 000837200 037__ $$aFZJ-2017-06179 000837200 041__ $$aEnglish 000837200 082__ $$a570 000837200 1001_ $$0P:(DE-Juel1)132000$$aGlück, Julian Marius$$b0$$eCorresponding author$$gmale$$ufzj 000837200 245__ $$aLigand interaction analysis of membrane-anchored proteins$$f- 99999 000837200 260__ $$aJülich$$bForschungszentrum Jülich GmbH Zentralbibliothek, Verlag$$c2010 000837200 300__ $$aVIII, 87 S : graph. Darst 000837200 3367_ $$2DataCite$$aOutput Types/Dissertation 000837200 3367_ $$0PUB:(DE-HGF)3$$2PUB:(DE-HGF)$$aBook$$mbook 000837200 3367_ $$2ORCID$$aDISSERTATION 000837200 3367_ $$2BibTeX$$aPHDTHESIS 000837200 3367_ $$02$$2EndNote$$aThesis 000837200 3367_ $$0PUB:(DE-HGF)11$$2PUB:(DE-HGF)$$aDissertation / PhD Thesis$$bphd$$mphd$$s1504004950_10814 000837200 3367_ $$2DRIVER$$adoctoralThesis 000837200 4900_ $$aSchriften des Forschungszentrums Jülich. Reihe Gesundheit / Health$$v31 000837200 502__ $$aUniversität Düsseldorf, Diss., 2010$$bDr.$$cUniversität Düsseldorf$$d2010 000837200 500__ $$aKeine Online-Freischaltung 000837200 520__ $$aDiverse cellular functions including signaling, transport, recognition and stability are performed by integral membrane proteins (IMPs). For this reason various studies focus on this prominent group of proteins. IMPs contain hydrophobic regions that restrict solubility in aqueous solutions. Unfortunately, many biophysical techniques require soluble analytes. Therefore, if IMPs are supposed to be studied they need to be maintained in a solubilized and functional state. This can be accomplished with different approaches that mimic the physicochemical properties of a membrane. In this work a novel membrane mimic termed nanodisc was applied for the study of an IMP. Nanodiscs are detergent-free model membranes that consist of ∼150 lipid molecules arranged as a bilayer. The discoidal lipid core is encircled by two copies of an amphipathic α-helical protein in a belt-like manner. A single disc possesses a diameter of ∼10 nm and a lipid-dependent thickness of ∼5 nm. The aim of the current thesis was the evaluation of nanodiscs as model membrane in structural and interaction analyses of IMPs. For this purpose a protocol enabling the successful incorporation of CD4mut, a truncated human CD4 (cluster of differentiation 4) receptor comprising transmembrane and cytoplasmic domains, was established. Structural studies by solution NMR spectroscopy on nanodisc embedded $^{13}$C/$^{15}$N-labeled CD4mut showed reasonably dispersed protein and lipid resonances in aliphatic $^{1}$H-$^{13}$C HSQC spectra. The $^{1}$H line widths are in the range of 15 to 30 Hz. In comparison with globular proteins of ∼150 kDa, similar to the molecular weight of a nanodisc, significantly broader lines are expected. These results demonstrated for the first time that IMPs in nanodiscs are amenable to liquid-state NMR spectroscopy. Furthermore, applicability of IMP containing nanodiscs as analytes in surface plasmon resonance (SPR) technology was examined. SPR measurements were conducted with an antibody directed against oligohistidines (His) immobilized on the sensor chip and either His-Ubiquitin or His-Ubiquitin fused to nanodisc-embedded CD4mut as analyte (loaded nanodisc). In both cases similar affinities (KD) close to 10 nM were determined. This is expected as both interactions should be governed by the same affinity between His-tag [...] 000837200 536__ $$0G:(DE-HGF)POF3-899$$a899 - ohne Topic (POF3-899)$$cPOF3-899$$fPOF III$$x0 000837200 588__ $$aDataset connected to Library of Congress 000837200 650_7 $$0(DE-588)4130026-9$$2gnd$$aMembranproteine 000837200 650_7 $$0(DE-588)4038575-9$$2gnd$$aMembrantransport 000837200 8564_ $$uhttp://www.gbv.de/dms/tib-ub-hannover/655087567.pdf 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.pdf$$yRestricted 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.gif?subformat=icon$$xicon$$yRestricted 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.jpg?subformat=icon-1440$$xicon-1440$$yRestricted 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.jpg?subformat=icon-180$$xicon-180$$yRestricted 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.jpg?subformat=icon-640$$xicon-640$$yRestricted 000837200 8564_ $$uhttps://juser.fz-juelich.de/record/837200/files/Gesundheit_31.pdf?subformat=pdfa$$xpdfa$$yRestricted 000837200 909CO $$ooai:juser.fz-juelich.de:837200$$pVDB 000837200 9131_ $$0G:(DE-HGF)POF3-899$$1G:(DE-HGF)POF3-890$$2G:(DE-HGF)POF3-800$$3G:(DE-HGF)POF3$$4G:(DE-HGF)POF$$aDE-HGF$$bProgrammungebundene Forschung$$lohne Programm$$vohne Topic$$x0 000837200 920__ $$lyes 000837200 9201_ $$0I:(DE-Juel1)VDB942$$kISB-3$$lStrukturbiochemie$$x0 000837200 980__ $$aphd 000837200 980__ $$aVDB 000837200 980__ $$abook 000837200 980__ $$aI:(DE-Juel1)VDB942 000837200 980__ $$aUNRESTRICTED