| dc.contributor.advisor | Kühnel, Karin Dr. | |
| dc.contributor.author | Behrens, Caroline Anna Julie | |
| dc.date.accessioned | 2014-08-06T08:42:51Z | |
| dc.date.available | 2014-08-06T08:42:51Z | |
| dc.date.issued | 2014-08-06 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0022-5F44-F | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-4638 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | |
| dc.subject.ddc | 571.4 | de |
| dc.title | Crystal Structure and Characterization of the SCOC Coiled Coil Domain | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Thumm, Michael Prof. Dr. | |
| dc.date.examination | 2013-08-07 | |
| dc.description.abstracteng | Autophagy is a conserved eukaryotic process for the degradation of cytosolic
content. An expanding double layered membrane takes up part of the cytoplasm
and matures to an autophagosomal vesicle. The autophagosome fuses
with the lysosome, where its content is degraded. The short coiled coil protein
(SCOC) forms a complex with fasciculation and elongation protein zeta 1
(FEZ1). Their complex is involved in autophagy regulation. Here, I present
the crystal structure of the functional important human SCOC coiled coil
domain (residues 78–159), which was determined at 2.8 Å resolution. SCOC
forms a parallel left handed coiled coil dimer. I observed two distinct dimers in
the crystal structure with a 3-molecule asymmetric unit, showing that SCOC
is conformational flexible. Importantly, several of the canonical hydrophobic
a/d-heptad core positions are occupied by well conserved, polar and charged
residues. I assumed that these non-canonical residues might have a strong
effect on the stability and oligomerization state of SCOC. The influence of
the residues was investigated by characterising mutant SCOC constructs. The
double core mutations E93V/K97L and N125L/N132V led to a change from
dimer to either trimer or tetramer formation and the thermostabilities of these
mutants were dramatically increased. Moreover, I present the formation of a
stable homogeneous complex of SCOC with the coiled coil domain of FEZ1.
A minimal FEZ1 region (residues 227–290) was sufficient for interaction with
SCOC. Complex formation with the SCOC double core mutants was impaired,
showing that dimerization of SCOC is essential for interaction with FEZ1. In
addition I identified SCOC surface residue R117 as important for binding.
The human pathogen Shigella flexneri escapes autophagy by a sophisticated
mechanism involving the Shigella flexneri proteins VirG and IcsB–IpgA. VirG
is an autotransporter protein, consisting of a signal sequence, a passenger
domain (residues 52–758) and a transmembrane domain. I established a purification
protocol for the VirG (52–758) under denaturing conditions. First
crystallization trials of refolded VirG passenger domain yielded spherulites
and microcrystalline structures. Initial studies of IcsB–IpgA indicate, that
IcsB contains flexible and presumably unfolded regions, while its chaperone
IpgA is well structured and stable. | de |
| dc.contributor.coReferee | Zweckstetter, Markus Prof. Dr. | |
| dc.contributor.thirdReferee | Rodnina, Marina Prof. Dr. | |
| dc.subject.eng | crystal structure SCOC | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0022-5F44-F-6 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 796307962 | |