Single molecule fluorescence reveals dynamic structures of SNARE protein assemblies

protein-protein interactions;synaptic vesicle;single molecule FRET;neurotransmitter release;membrane fusion

Choi, Ucheor B. ; Alex Smirnov, Committee Member,Celeste Sagui, Committee Member,Robert Riehn, Committee Member,Keith Weninger, Committee Chair,Choi, Ucheor B. ; Alex Smirnov ; Committee Member ; Celeste Sagui ; Committee Member ; Robert Riehn ; Committee Member ; Keith Weninger ; Committee Chair

University:North Carolina State University

关键词: protein-protein interactions;    synaptic vesicle;    single molecule FRET;    neurotransmitter release;    membrane fusion;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/6220/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Conformational information about proteins can often reveal the mechanisms of their biological functions. This thesis examines conformational aspects of the synaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein complex that is essential for membrane fusion leading to Ca2+ triggered neurotransmitter release. Biochemical and high-resolution structural studies of SNARE proteins and several different assemblies of these proteins have provided a foundation for our understanding of neurotransmitter release, but the exact mechanisms these protein machines use to effect membrane fusion are still unclear. Here we apply single molecule fluorescence spectroscopy to this problem.Single molecule fluorescence resonance energy transfer (smFRET) is used to characterize dynamic aspects of intermediate structures of SNARE proteins along the pathway to full SNARE complex formation.Several SNAREs fall into the class of intrinsically unstructured proteins.We have used FRET to characterize these unstructured molecules as well as the development of specific conformations as they assemble into the SNARE complex.Finally, the structure of synaptotagmin bound to the fully assembled SNARE complex is derived using smFRET measurements as constraints for docking calculations. A common theme is the dynamic nature of many of these proteins and complexes. Dynamic protein structures are increasingly being recognized as critical for many functions of a wide variety of proteins.The methods developed in this thesis are expected to find increasing applications in future studies of the structure-function relationship in many biological macromolecules.