Spezifität der Wechselwirkung von Collybistin 2 mit Phosphatidylinositolphosphaten: Einfluss der verschiedenen Proteindomänen

Ludolphs, Michaela

dc.contributor.advisor	Steinem, Claudia Prof. Dr.
dc.contributor.author	Ludolphs, Michaela
dc.date.accessioned	2015-05-27T08:18:12Z
dc.date.available	2015-10-27T23:50:05Z
dc.date.issued	2015-05-27
dc.identifier.uri	http://hdl.handle.net/11858/00-1735-0000-0022-5FF8-C
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-5091
dc.language.iso	deu	de
dc.publisher	Niedersächsische Staats- und Universitätsbibliothek Göttingen	de
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/3.0/
dc.subject.ddc	540	de
dc.title	Spezifität der Wechselwirkung von Collybistin 2 mit Phosphatidylinositolphosphaten: Einfluss der verschiedenen Proteindomänen	de
dc.type	doctoralThesis	de
dc.title.translated	Specificity of collybistin interaction with phosphoinositides: Impact of the individual protein domains	de
dc.contributor.referee	Steinem, Claudia Prof. Dr.
dc.date.examination	2015-04-27
dc.description.abstracteng	Collybistin, a brain-specific GEF, is required for gephyrin clustering at the postsynaptic membrane. This is important for the correct arrangement of GABAA and glycine receptors. Binding of collybistin to a membrane is initiated by a C-terminal PH-domain. In general, this domain is known to specifically bind to phosphoinositides (PIPs) in the membrane. The aim of this study was to design an in vitro assay that enables detailed studies of binding of collybistin binding to phosphoinositides. With this assay we analyzed the impact of different collybistin 2 domains on binding to a membrane. Solid-supported lipid bilayers (SLB) with different phosphoinositides mimicking the cellular postsynaptic membrane. This approach allowed to investigate specific binding of the protein isoforms using reflective interference spectroscopy (RIfS). We found out that the PH domain (CB2PH) from collybistin prefers binding to monophosphorylated PIPs with moderate binding affinity. The PH/DH-tandem domain (CB2SH3-) reveals a weaker binding affinity to different PIPs except for PI(3,5)P2. In addition the collybistin isoform containing an additional SH3 domain (CB2SH3+) showed no significant binding to PIPs. This suggested a closed/inactive protein conformation. Activation of CB2SH3+ is initiated in vivo by the postsynaptic cell adhesion protein Neuroligin-2. For our in vitro studies this was successfully mimicked by two point mutations in the protein isoform CB2SH3+/W24A-E262A. In contrast to CB2SH3+, the partly-activated mutant CB2SH3+/W24A-E262A binds to phosphoinositides in SLBs with decreasing binding affinity to PI(3)P, PI(4)P and PI(3,5)P2. This study provides new insights into the binding of collybistin to phosphoinositides in membranes. In order to draw a comprehensive picture of this protein to membrane binding process, the impact of each protein domain was investigated.	de
dc.contributor.coReferee	Köster, Sarah Prof. Dr.
dc.subject.eng	Collybistin	de
dc.subject.eng	Phosphoinositides	de
dc.subject.eng	solid supported membranes	de
dc.subject.eng	PIP	de
dc.subject.eng	SH3-domain	de
dc.subject.eng	DH-domain	de
dc.subject.eng	PH-domain	de
dc.subject.eng	reflectometric interference spectroscopy	de
dc.subject.eng	protein purification E.coli	de
dc.identifier.urn	urn:nbn:de:gbv:7-11858/00-1735-0000-0022-5FF8-C-1
dc.affiliation.institute	Fakultät für Chemie	de
dc.subject.gokfull	Chemie (PPN62138352X)	de
dc.description.embargoed	2015-10-27
dc.identifier.ppn	826213863

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