Chemistry Reference
In-Depth Information
However, the fusion rate is slower due to the reduced diffusion of the liposomes (due
to the higher viscosity of the buffer as described above). Electrostatic repulsion at
low ionic strength therefore increases the energy barrier of membrane fusion.
As discussed earlier, according to the current picture of SNARE-mediated fusion,
the SNARE nucleation occurs even before any action of
Ca
2
+
or synaptotagmin-1.
We therefore investigated if this is still true when there are repulsive interactions
between the liposomes, when the ionic strength of the buffer is lowered. This is done
by tagging the complementary SNARE proteins by FRET pair fluorophores rather
than the lipids in a vesicle as shown in the schematic in Fig.
3.8
. It can be seen in the
bottom panel of Fig.
3.8
that SNARE nucleation does not occur when electrostatic
repulsion between the vesicles is high.When the buffer contains 150mMKCl (normal
ionic strength), the donor fluorescence reduces significantlywith time, indicating that
the SNARE proteins come close enough to nucleate and form a coil-coil structure.
However, when the buffer salt concentration is reduced to 5mM KCl (low ionic
strength), SNARE nucleation does not take place. Further, the addition of 1mM
Ca
2
+
to this solution did not cause any SNARE-nucleation either.
In the absence of liposomes that house the SNARE proteins, it can be checked if
SNARE nucleation itself is affected by electrostatic interactions. SNARE nucleation
is not expected to be affected since the interactions are not mediated by charges.
Indeed, when we repeat the above experiment, now without the R-SNARE or the
Q-SNARE respectively, SNARE nucleation does proceed. In the first case (R
sol
),
the fluorescent tagged synaptobrevin-2 is present in solution, while the SNAP25 and
Fig. 3.8
SNARE nucleation is blocked at low ionic buffer strengths.
Left panel
When the fluo-
rescently labelled SNARE proteins are present in their respective liposomes, reducing the ionic
strength leads to a complete blocking of SNARE nucleation. The addition of 1mM
Ca
2
+
does
not change the situation.
Right panel
In the absence of the repulsive interactions caused due to the
lipids in the liposomes, SNARE nucleation proceeds normally irrespective of the ionic strength of
the buffer used
