Fluorescence Correlates of Single Vesicles to Analyze the Behavior of Membrane-manipulating Proteins

by Tobias Grothe
Doctoral thesis
Date of Examination:2024-09-26
Date of issue:2025-01-24
Advisor:Prof. Dr. Peter Jomo Walla
Referee:Prof. Dr. Peter Jomo Walla
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Reinhard Jahn
Persistent Address: http://dx.doi.org/10.53846/goediss-11041

 

 

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Abstract

English

Soluble NSF-attachment protein receptors (SNAREs) are integral to eukaryotic membrane fusion, providing the necessary energy to surmount the repulsive forces between two opposing membranes. This is achieved through the formation of a four-helix bundle, which exhibits significant hysteresis. Subsequently, the AAA ATPase NSF and soluble NSF adaptor proteins (SNAPs) facilitate their reassembly, recharging the system. While substantial knowledge has been amassed regarding the pre-fusion stage, the post-fusion phase is only now coming to light, thanks to recent publications that provide structural insights into the disassembly apparatus involving NSF and α-SNAP. Nevertheless, numerous questions remain unanswered about both stages, including the precise initiation and regulatory mechanisms of fusion, as well as the processes and consequences of disassembly. Most of our current understanding stems from studies on soluble or truncated SNARE proteins, which may overlook essential mechanisms, particularly concerning the nascent post-fusion phase. This research endeavors to bridge some of these gaps by constructing a fully membrane-anchored SNARE complex and utilizing fluorescence cross-correlation spectroscopy (FCCS) to monitor disassembly on a two-dimensional membrane. The findings suggest a fragile balance between disassembly and reassembly when NSF is in limited supply. Additionally, the study reveals a relatively rapid reassembly of SNAREs immediately following disassembly. Moreover, this work characterizes the binding of α-SNAP to protein-free membranes. Despite its modest affinity, the measured binding strength implies near-complete association under physiological conditions.
Keywords: Fluorescence Cross-Correlation Spectroscopy; SNARE; GUV; lipid membrane; SNAP-25; Syntaxin-1A; Synaptobrevin 2
 
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