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dc.contributor.advisor Ficner, Ralf Prof. Dr.
dc.contributor.author Meese, Sandra
dc.date.accessioned 2015-08-27T08:29:26Z
dc.date.available 2015-08-27T08:29:26Z
dc.date.issued 2015-08-27
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-0022-6089-2
dc.language.iso eng de
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc 570 de
dc.title Biochemical studies of the synaptic protein otoferlin de
dc.type doctoralThesis de
dc.contributor.referee Ficner, Ralf Prof. Dr.
dc.date.examination 2015-02-17
dc.description.abstracteng The C2 domain containing protein otoferlin is required for a late step in exocytosis from auditory inner hair cells and was hypothesized to act as a synaptic Ca2+ sensor. More roles for otoferlin in hair cell synaptic function have been proposed but have just begun being studied. To address the questions for potential interaction partners of otoferlin or its overall structure several C2 domain containing protein fragments were expressed and purified from E. coli. The known ability of C2 domains to bind Ca2+ was tested using MicroScale Thermophoresis (MST) and revealed no indication for Ca2+ binding within the first three C2 domains of the protein while the otoferlin C2F domain gave a distinct signal for Ca2+ binding. Additionally the roles of the present aspartate residues within the binding region were analyzed, revealing that mutations in these aspartate residues reduced Ca2+ binding to a similar degree. It seems that in all cases the structure is affected in a comparable way, leading to a 10 fold increase in the Kd. Further tested mutations known to affect hearing showed a similar decrease in Ca2+ binding. In order to identify phosphorylation sites in the protein two long otoferlin fragments were incubated with CaMKIIδ which resulted in ten phosphorylation sites. Ca2+ binding studies revealed that the incorporated aspartate residues mimicking phosphorylation sites within the C2C and C2F domain of the protein affect the Ca2+ binding affinity in different ways. For the C2C domain an increase in the affinity to bind Ca2+ was found whereas the incorporated aspartate residues within the C2F domain led to a ten fold decreased affinity. Pull down experiments were performed to get information about potential interaction partners of otoferlin which are assumed to play a role within the unknown synaptic mechanism of otoferlin action. Several of the identified interacting proteins are involved in endocytosis, exocytosis or trafficking processes. In ongoing studies these potential interaction partners are currently validated. Thermal shift experiments of the purified otoferlin C2 domains displayed that the melting temperature of a C2 domain can be increased either in presence of Ca2+ or neighboring C2 domains. This reveals that the Ca2+ binding in some C2 domains probably play an essential role in the function of the protein whereas complex forming of two or three C2 domains increases the stability of the whole protein. In addition the C2ABC I515T mutant displayed a decrease in thermal stability in comparison to the wild type fragment, giving a molecular mechanism explaining how the I515T mutation leads to temperature sensitive deafness in humans. For further analysis regarding the overall structure of the protein, crystals were obtained containing the first three C2 domains. Solving the structure is still an ongoing project, as it turned out that the structure cannot be solved by molecular replacement. de
dc.contributor.coReferee Moser, Tobias Prof. Dr.
dc.subject.eng Otoferlin de
dc.subject.eng C2-domain de
dc.identifier.urn urn:nbn:de:gbv:7-11858/00-1735-0000-0022-6089-2-5
dc.affiliation.institute Biologische Fakultät für Biologie und Psychologie de
dc.subject.gokfull Biologie (PPN619462639) de
dc.identifier.ppn 834026902

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