Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) realize a central task in exocytotic and vacuolar trafficking pathways by implementing compartment-specific membrane fusion processes.Different from the bona fide type Ⅱ majority of SNARE-proteins containing a proteinaceous membrane linkage the chosen Ykt6 cycles between an inactive cytosolic and an active membrane-anchor-associated state.Starting from the closed cytosolic farnesylated condition a palmitoylation of the Ykt6 leads to a conformational change resulting in a stable membrane attachment,which is controlled by the interacting of the N-terminal domain and the SNARE motif influenced by the presence of shielding lipids.Single-molecule F(o)rster Resonance Energy Transfer (smFRET) combined with Fluorescence Correlation Spectroscopy (FCS) asserts as powerful techniques depicting intramolecular time-dependent conformational changes.Two-dye-labeling of proteins enable probing of distribution of conformational states.Furthermore,Auto-and Crosscorrelation analysis allow us to investigate protein dynamics on different time scales.In this work,we double-labeled a rat rYkt6-E175C-mutant with a suitable FRET pair to study the conformational state distribution in a vesicle-based smFRET-Setup and to observe confonnational dynamics between inactive and active states in a Fluorescence Cross Correlation Spectroscopy Setup (FCCS).