Passage
Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1) .
Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer
An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF) , which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2.
Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin
Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5) :2213-7.
-During cis-complex disassembly, what consequence of NSF function is most likely responsible for destabilization of the cis-complex?

Question

Passage
Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1) .

Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer

An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF) , which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2.

Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin
Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5) :2213-7.
-During cis-complex disassembly, what consequence of NSF function is most likely responsible for destabilization of the cis-complex?

Quizplus · Accepted Answer

11ecba2d_0dbd_0a19_a3bf_bb8d5dccb503_MD0008_00 Typically, hydrophilic amino acids (polar and charged) are found on the outside of proteins, where they interact favorably with water molecules. Hydrophobic amino acids are usually shielded from the solvation layer in the protein interior or found at protein-protein interfaces. This arrangement is entropically favorable, as water molecules cannot form hydrogen bonds with hydrophobic residues, and therefore must organize around these residues. Leucine is hydrophobic, and leucine zipper layers act as a watertight seal around the zero ionic layer, in which a positively charged, basic arginine (R) residue interacts with polar glutamine (Q) residues at the interface of the complexed proteins. NSF destabilizes the cis-complex by breaking the leucine zipper seal (unzipping). Without the watertight seal provided by the leucine zipper, the charged and polar residues of the zero ionic layer are exposed to water. Because water is polar, exposure of the charged and polar residues results in preferential binding to water and resultant destabilization of the cis-complex. (Choice B) The charges on residues in the zero ionic layer do not change, so no additional repulsive forces are added by breaking the leucine zipper. (Choice C) A leucine zipper is a common protein motif composed of two amphipathic α-helices, meaning they possess both hydrophilic and hydrophobic residues. Unzipping would not change the residues, and therefore would not change the amphipathic qualities of the zipper. (Choice D) Release of v-SNARE proteins would not occur until after cis-complex disassembly. Educational objective: Hydrophilic amino acids are typically found on the outside of proteins whereas hydrophobic amino acids exist in the protein interior and protein-protein interfaces. When polar or charged amino acids are exposed to water, they will preferentially form hydrogen bonds with water molecules.

Passage Presynaptic Nerve Terminals Release Neurotransmitters Via Synaptic Vesicle Exocytosis. This