Design and Synthesis of Modified SNARE Proteins with Respect to the ¿¿SNAP/NSF Mediated Disassembly

Soluble N-ethylmaleimide-sensitive attachment receptor (SNARE) proteins are the

key players in membrane fusion. Localized in opposed membranes, they assemble via

the SNARE motif in a stable four-helix bundle bringing the membranes close to each

other and promoting membrane fusion by using the energy release during complex

formation. SNARE complex assembly is regulated by several proteins. One of these,

Complexin, is known to partially associate with the core complex, it may stabilize

SNARE complex intermediates and unbinds upon calcium trigger. Nevertheless, the

exact function of Complexin is still under discussion. After membrane fusion the

recycling of free SNARE proteins is mediated by the AAA+ protein NSF in conjunction

with its cofactor a-SNAP. Afterwards, the individual SNARE proteins are available for

another round of membrane fusion.

To date, no effective model systems for preventing or at least decelerating the

disassembly mechanism are known. Development of a potent inhibitor of the a-

SNAP/NSF mediated disassembly was carried out. Therefore, the SNARE motif of

Synaptobrevin, one of the SNARE proteins, was used as a model system for the

investigation of defined SNARE/SNAP complex recognition sites. The full length of the

SNARE motif of Synaptobrevin was obtained using solid phase peptide synthesis.

Different modifications at various residues within the sequence were introduced in

order to identify important interactions between a-SNAP and the SNARE complex and

to 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-SNAP

recognition through enhanced interaction between the ß-mimic and the

Synaptobrevin and Syntaxin helices. By development of the Complexin ß-peptide

mimic as a 14-helix, the advantages of a well-defined secondary structure with high

helix propensity are obtained. Furthermore, the binding fragment of Complexin was

performed as a-peptide, extended with amino acids known to promote the a-helical

propensity.

For understanding of biological systems the investigation of conformational dynamics

and interactions of individual proteins is important. Therefore, in a final part, small

independently folding protein domains were synthesized by solid phase peptide

synthesis and labeled with respect to the development of the single molecule

fluorescence spectroscopy (smFRET) technique. This method is a convenient tool of

monitoring single folding and unfolding events of proteins.