Proteintransport in E. coli: Untersuchung der Konformationsänderungen des Chaperons SecB durch Bindung an das Modellsubstrat Bovine Pancreatic Trypsin Inhibitor (BPTI) mit Hilfe der EPR-Spektroskopie

  • SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

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Verfasserangaben:Michaela Haimann
URN (Permalink):urn:nbn:de:hbz:386-kluedo-29432
Betreuer:Wolfgang E. Trommer
Sprache der Veröffentlichung:Deutsch
Veröffentlichungsdatum (online):14.03.2012
Jahr der Veröffentlichung:2012
Veröffentlichende Institution:Technische Universität Kaiserslautern
Titel verleihende Institution:Technische Universität Kaiserslautern
Datum der Annahme der Abschlussarbeit:08.03.2012
Datum der Publikation (Server):15.03.2012
Fachbereiche / Organisatorische Einheiten:Fachbereich Chemie
DDC-Sachgruppen:5 Naturwissenschaften und Mathematik / 540 Chemie
5 Naturwissenschaften und Mathematik / 570 Biowissenschaften, Biologie
Lizenz (Deutsch):Standard gemäß KLUEDO-Leitlinien vom 15.02.2012