guest ::
| Home > Publications database > Ligand interaction analysis of membrane-anchored proteins |
| Home > Publications database > Ligand interaction analysis of membrane-anchored proteins |
| Book/Dissertation / PhD Thesis | FZJ-2017-06179 |
2010
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-89336-683-5
Abstract: Diverse 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 [...]
Keyword(s): Membranproteine ; Membrantransport
|
The record appears in these collections: |
PDF
PDF (PDFA)