The Perception of Hydrophobic Clusters in the Native and Partially Unfolded States
219
4.3
Equilibrium Binding of bis-ANS to Native GLA
We determined the binding properties ofbis-ANS to native GLA in the previously
selected conditions (4°C and 2 mM EGTA or 37°C and 2 mM Ca 2 +) by performing
two series of fluorescence titrations, as described in Materials and Methods.
The strong fluorescence increase at the start of the titrations of 1 !!M bis-ANS
with apo-GLA and 1 !!M apo-GLA with bis-ANS (Fig.s 15.5 A and B, filled
squares) are characteristic for the strong binding of (one or more) fluorophore
molecules to the Ca 2 +-free protein. As can be expected for such a strong binding,
the fluorescence increase for the addition of bis-ANS to apo-GLA (Fig. 15.5 B,
filled squares) readily tends to a limit. However, this limit is not obtained by the
addition of apo-GLA to bis-ANS (Fig. 15.5 A, filled squares). It is judged that the
most plausible explanation for this continued fluorescence increase at higher
Fig. 15.4. Thermal transition curves of
GLA measured by the ellipticity change at
270 nm. Conditions: 25 f-IM GLA in TrisHCI buffer containin~ 2 mM EGTA (ftlled
squares) or 2 mM Ca + (open squares), after
equilibration with 60 f-IM bis-ANS in 2 mM
EGTA (filled circles), containing 3.3 photolabeled bis-ANS molecules per GLA in
2 mM Ca2+ (open circles) (Vanderheeren
et al. 1998)
Fig. 15.5. Fluorescence titrations of 1 f-IM
bis-ANS with GLA (A) and 1 f-IM GLA with
bis-ANS (B). Scatchard plots (C) and Hill
plots (D) for the titration of 1 f-IM GLA with
bis-ANS. Conditions: 4°C and 2 mM EGTA
(filled squares) or 37°C and 2 mM Ca 2 +
(open squares) (Vanderheeren et al. 1998)
o
E
"0
'" E o
Ol)
d)
"0
"--"
o
r'"
~
l\1
C/}
Z
~
'"
:0
OJ
OJ
.!:
:0
C!
15
J;)
I
0 " ... .... .. ..
' .. f···
-1 00
-200
-300
~
.,.•
•
•
•
•
•
COCD 0
I
c
~
rP~
-400 0 '::--'-~-'---'-::---'--~-'----:!-=-~.
20
40
60
80
100
temperature (0C)
200
A
B
200
•
CD
,.,.
. 0
L\1
• 0
100
•
100
0
0
0
150 300 0
150 300
rGL 1
[bis-A S]
(~ \'f )
I~ 1)
r ~
1.5
C
\
0• \
0.0 ~
• II
' "
cP 00 \ Do
.=.,
-1.5 2:
2
4
-5
-4
log fbi -
S]