|
Figure 7.
Figure 7. The Synaptotagmin 1 C[2]B-Domain Binds
PS-Containing Vesicles in a Ca^2+-Dependent Manner(A) and (B)
show FRET measurements from the C[2]A-domain (A) and
C[2]B-domain (B) to phospholipid vesicles containing 10%
dansyl-PE, 25% PS, and 65% PC in the presence of 0.5 mM EDTA
(red traces) or 0.2 mM Ca^2+ (black traces). The protein
concentration was 1 Ž§¼M and the lipid concentration was 0.022
mg/ml. In (B), the green trace was acquired with wild-type
C[2]B-domain in 1 mM Mg^2+ and the blue trace with the D309N
mutant C[2]B-domain in the presence of 0.2 mM Ca^2+. A spectrum
acquired under identical conditions but without protein was
subtracted for each data set. (C) and (D) show Ca^2+ dependence
of phospholipid binding measured by FRET for the C[2]A-domain
(C) and the C[2]B-domain (D). The relative change in FRET
measured as in (A) and (B) is represented as a function of the
Ca^2+ concentration for the C[2]A-domain (C) and the
C[2]B-domain (D). The data represent an average of three
measurements which yielded an apparent Ca^2+ affinity and Hill
coefficient of 54 Ž§¼M Ca^2+ and 1.3, respectively, for the
C[2]A-domain, and 48 Ž§¼M Ca^2+ and 1.6, respectively, for the
C[2]B-domain. (E) shows Ca^2+-dependent phospholipid binding to
the C[2]A-domain and the C[2]B-domain monitored by
centrifugation. Samples containing purified GST-C[2]A-domain or
GST-C[2]B-domain were incubated with liposomes (PS/PC 25:75) and
various Ca^2+ concentrations as indicated. The samples were
centrifuged and, after washing the precipitated liposomes, bound
protein was analyzed by SDS-PAGE and Coomassie Blue staining.
(F) and (G) show Ca^2+-dependent phospholipid binding to
immobilized GST-C[2]A-domain (F) and GST-C[2]B-domain (G). GST
fusion proteins reattached to glutathione-agarose after
purification in solution were incubated with ^3H-labeled
liposomes (PS/PC 30:70) at various Ca^2+ concentrations and
after washing the resin, the bound lipids were measured by
scintillation counting. Each graph shows a representative
experiment performed in triplicate. The average apparent Ca^2+
affinity and Hill coefficient obtained from four independent
experiments was 13 Ž§¼M Ca^2+ and 3.6, respectively, for the
C[2]A-domain, and 15 Ž§¼M Ca^2+ and 1.7, respectively, for the
C[2]B-domain. Some error bars are not visible because of their
small size.
|
