UNDERSTANDING CELL SECRETION AND MEMBRANE FUSION PROCESSES ON THE NANOSCALE USING THE ATOMIC FORCE MICROSCOPE - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 110

calcium [Ca(H 2 O) n ] 2+ , however, with a hydration shell having six water
molecules and measuring ~6 Å would be excluded from the t-/v-SNARE-
apposed inter-bilayer space. Hence, calcium has to be present in the buffer
solution when t-SNARE vesicles and v-SNARE vesicles meet. Indeed, studies
demonstrate that if t-SNARE and v-SNARE vesicles are allowed to mix in a
calcium-free buffer, there is no fusion following post addition of calcium. 22
How does calcium work? Calcium bridging of apposing bilayers may
lead to the release of water from the hydrated Ca 2+ ion, leading to bilayer
destabilization and membrane fusion. Additionally, the binding of calcium
to the phosphate head groups of the apposing bilayers may also displace the
loosely coordinated water at the PO-lipid head groups, resulting in further
dehydration, leading to destabilization of the lipid bilayer and membrane
fusion. Recent studies in the laboratory, 23 using molecular dynamics
simulations in the isobaric-isothermal ensemble to determine whether
Ca was capable of bridging opposing phospholipid head groups in the

early stages of the membrane fusion process, indeed demonstrate this to
be the case. Furthermore, the distance between the oxygen atoms of the
opposing PO-lipid head groups bridged by calcium was found to be 2.92 Å,
in agreement with the 2.8 Å distance previously determined using X-ray
diffraction measurements. The hypothesis that there is loss of coordinated
water both from the hydrated calcium ion and oxygen of the phospholipid
head groups in opposing bilayers, following calcium bridging, is further
demonstrated from the study.

2+

23
In the presence of ATP, the highly stable, membrane-directed and self-
assembled t-/v-SNARE complex can be disassembled by a soluble ATPase,
the Careful examination of

the partially disassembled t-/v-SNARE bundles within the complex using
AFM demonstrates a left-handed super coiling of SNAREs. These results
demonstrate that t-/v-SNARE disassembly requires the right-handed
uncoiling of each SNARE bundle within the ring complex, demonstrating NSF
to behave as a right-handed molecular motor. 26 Using circular dichroism (CD)
spectroscopy, we reported 27 for the irst time that both t-SNAREs and v-SNARE
and their complexes in buffered suspension exhibit deined peaks at CD signals
of 208 and 222 nm wavelengths, consistent with a higher degree of helical
secondary structure. Surprisingly, when incorporated in lipid membrane,
both SNAREs and their complexes exhibit reduced folding. Furthermore,
these studies demonstrated that NSF, in the presence of ATP, disassembles the
SNARE complex as relected from the CD signals demonstrating elimination
of α-helices within the structure. These results demonstrate that NSF-ATP is
suficient for the disassembly of the t-/v-SNARE complex. These studies 20-27
have provided a molecular understanding of SNARE-induced membrane
fusion in cells.

N -ethylmaleimide-sensitive factor (NSF).

25-27