Academic Journal
أعرض في EDS

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

التفاصيل البيبلوغرافية
العنوان: Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements
المؤلفون: Laugwitz, Jeannette M., Haeri, Haleh H., Kaiser, Anette, Krug, Ulrike, Hinderberger, Dariush, Beck-Sickinger, Annette G., Schmidt, Peter
Publication Status: publishedVersion
بيانات النشر: MDPI, 2020.
سنة النشر: 2020
المجموعة: Hochschulschriftenserver (HSSS) der SLUB Dresden
Original Material: urn:nbn:de:bsz:15-qucosa2-848429
مصطلحات موضوعية: GPCR, Y2R, EPR, DEER, refolding, nitroxide spin labels, MTSL, IDSL, info:eu-repo/classification/ddc/540, ddc:540
الوصف: The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.
Original Identifier: oai:qucosa:de:qucosa:84842
نوع الوثيقة: Article
اللغة: English
تدمد: 1420-3049
Relation: 4143
الاتاحة: https://ul.qucosa.de/id/qucosa%3A84842
https://ul.qucosa.de/api/qucosa%3A84842/attachment/ATT-0/
Rights: info:eu-repo/semantics/openAccess
رقم الانضمام: edsndl.DRESDEN.oai.qucosa.de.qucosa.84842
قاعدة البيانات: Networked Digital Library of Theses & Dissertations
الوصف
تدمد:14203049