Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex

被引:80
作者
Bowen, ME
Weninger, K
Ernst, J
Chu, S
Brunger, AT
机构
[1] Stanford Univ, Howard Hughes Med Inst, Stanford, CA 94305 USA
[2] Stanford Univ, Dept Cellular & Mol Physiol, Stanford, CA 94305 USA
[3] Stanford Univ, Dept Neurol, Stanford, CA 94305 USA
[4] Stanford Univ, Dept Neurol Sci, Stanford, CA 94305 USA
[5] Stanford Univ, Stanford Synchrotron Radiat Lab, Stanford, CA 94305 USA
[6] Stanford Univ, Dept Phys, Stanford, CA 94305 USA
基金
美国国家卫生研究院; 美国国家航空航天局; 美国国家科学基金会;
关键词
D O I
10.1529/biophysj.104.054064
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler'' to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.
引用
收藏
页码:690 / 702
页数:13
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