Bøger af Annika Groschner

Soluble N-ethylmaleimide-sensitive attachment receptor (SNARE) proteins are thekey players in membrane fusion. Localized in opposed membranes, they assemble viathe SNARE motif in a stable four-helix bundle bringing the membranes close to eachother and promoting membrane fusion by using the energy release during complexformation. SNARE complex assembly is regulated by several proteins. One of these,Complexin, is known to partially associate with the core complex, it may stabilizeSNARE complex intermediates and unbinds upon calcium trigger. Nevertheless, theexact function of Complexin is still under discussion. After membrane fusion therecycling of free SNARE proteins is mediated by the AAA+ protein NSF in conjunctionwith its cofactor a-SNAP. Afterwards, the individual SNARE proteins are available foranother round of membrane fusion.To date, no effective model systems for preventing or at least decelerating thedisassembly mechanism are known. Development of a potent inhibitor of the a-SNAP/NSF mediated disassembly was carried out. Therefore, the SNARE motif ofSynaptobrevin, one of the SNARE proteins, was used as a model system for theinvestigation of defined SNARE/SNAP complex recognition sites. The full length of theSNARE motif of Synaptobrevin was obtained using solid phase peptide synthesis.Different modifications at various residues within the sequence were introduced inorder to identify important interactions between a-SNAP and the SNARE complex andto prevent a-SNAP recognition.Additionally, the regulator protein Complexin was synthesized as a ß-peptide analog,also designed to inhibit the disassembly mechanism by preventing a-SNAPrecognition through enhanced interaction between the ß-mimic and theSynaptobrevin and Syntaxin helices. By development of the Complexin ß-peptidemimic as a 14-helix, the advantages of a well-defined secondary structure with highhelix propensity are obtained. Furthermore, the binding fragment of Complexin wasperformed as a-peptide, extended with amino acids known to promote the a-helicalpropensity.For understanding of biological systems the investigation of conformational dynamicsand interactions of individual proteins is important. Therefore, in a final part, smallindependently folding protein domains were synthesized by solid phase peptidesynthesis and labeled with respect to the development of the single moleculefluorescence spectroscopy (smFRET) technique. This method is a convenient tool ofmonitoring single folding and unfolding events of proteins.