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Biophysical analysis of protein-protein and protein-small molecule interactions

dc.contributor.advisorFamulok, Michael
dc.contributor.authorAlbertoni, Barbara
dc.date.accessioned2020-04-17T07:47:32Z
dc.date.available2020-04-17T07:47:32Z
dc.date.issued29.11.2011
dc.identifier.urihttps://hdl.handle.net/20.500.11811/5044
dc.description.abstractThe validation of small molecule inhibitors identified by high throughput screening requires a set of robust assays for interaction analysis. Here I describe the implementation of three methods: bioluminescence resonance energy transfer (BRET) for the analysis of protein/protein interaction in cells, surface plasmon resonance (SPR) for the measurement of binding kinetics in vitro and capture compound mass spectrometry (CCMS) for the determination of binding specificity in proteome.
All the methods described were tested on cytohesins, a family of guanine nucleotide exchange factors. The very recent discovery of their additional role in the regulation of receptor tyrosine kinases (RTKs) signalling and the availability of specific small molecule inhibitors (the Secins) made them an interesting target.
BRET was applied to the analysis of a possible binding of the cytohesin ARNO to the RTK EGF receptor (EGFR). Two strategies were devised: a direct, where the interaction of ARNO and EGFR was monitored, and an indirect, which followed the changes in the EGFR/EGFR interaction upon overexpression of ARNO.
Two SPR approaches were developed to analyse the interaction between ARNO and the EGFR on the one hand, and to determine the kinetic parameters of binding of ARNO to its inhibitor Secin16 on the other hand.
For CCMS, a photoreactive affinity based SecinH3 probe (SecinH3-TPD) was synthesised and its ability to specifically label ARNO was shown, although the labelling yield was limited by low solubility. A protocol for the enrichment, digestion and MS-analysis of the labelled proteins was established.
en
dc.language.isoeng
dc.rightsIn Copyright
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.subjectOberflächenplasmonresonanz
dc.subjectBiolumineszenz-Resonanz-Energie-Transfer
dc.subjectSurface plasmon resonance
dc.subjectCapture compound mass spectrometry
dc.subjectAffinity based protein profiling
dc.subjectCytohesin
dc.subjectSecinH3
dc.subject.ddc540 Chemie
dc.subject.ddc570 Biowissenschaften, Biologie
dc.titleBiophysical analysis of protein-protein and protein-small molecule interactions
dc.typeDissertation oder Habilitation
dc.publisher.nameUniversitäts- und Landesbibliothek Bonn
dc.publisher.locationBonn
dc.rights.accessRightsopenAccess
dc.identifier.urnhttps://nbn-resolving.org/urn:nbn:de:hbz:5n-26661
ulbbn.pubtypeErstveröffentlichung
ulbbnediss.affiliation.nameRheinische Friedrich-Wilhelms-Universität Bonn
ulbbnediss.affiliation.locationBonn
ulbbnediss.thesis.levelDissertation
ulbbnediss.dissID2666
ulbbnediss.date.accepted20.10.2011
ulbbnediss.fakultaetMathematisch-Naturwissenschaftliche Fakultät
dc.contributor.coRefereeMayer, Günter


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