Abstract
Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.
Highlights
Previous studies claimed a potential model such that single-lipidated Ykt[6] exists in the cytosol and adopts an auto-inhibited conformation via farnesyl-dependent interaction between its sensitive factor attachment protein receptors (SNARE) core and longin domain[18]
We explored the conformational distributions and dynamics using single-molecule Förster Resonance Energy Transfer[27,28,29,30,31]
Judging from the size of longin domain and the SNARE core of Ykt[6], the conformational dynamics of this intramolecular movement may be in the sub-millisecond range
Summary
Previous studies claimed a potential model such that single-lipidated (i.e. farnesylation at Cys195) Ykt[6] exists in the cytosol and adopts an auto-inhibited conformation via farnesyl-dependent interaction between its SNARE core and longin domain[18]. To the best of our knowledge, the conformations of Ykt[6] in these in vivo enzymolysis studies were mostly inferred from comparison of mutants with wild type proteins[15,18] These methods can neither observe the conformational change nor measure the dynamics directly. We aim to conduct quantitative measurements of the conformational dynamics of rYkt[6] and provide a benchmark result for theoretical studies of Ykt[6] functional principles For this purpose, single molecule fluoresence methods are powerful means to investigate SNARE systems at molecular level. With increasing DPC, Ykt[6] appears to reside mostly in one of the conformational states It represents a coordinated action of the longin domain, the SNARE core, and the attached lipids. The Ykt6/DPC results reflect that when Ykt[6] is regulated by DPC, it would prefer a stable and rigid state possibly facilitating the formation of a SNARE complex
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