Chemistry Reference
In-Depth Information
Figure 13.9 Probing protein chemistry (A) A
disulfide-bonded protein. An engineered
disulfide bond is introduced between two
residues in the protein (yellow). This results in 46
unsequestered residues (red) and 43
sequestered residues (green) (B) Applying a
mechanical force first triggers the unfolding and
extension
unsequestered unfolding DL
1
(10.5 nm) from
disulfide reduction events DL
2
(14.2 nm) in the
presence of DTT (D) Force sensitivity and bond
lengthening in the thiol/disulfide exchange
chemical reaction: a semi-logarithmic plot of the
rate of thiol/disulfide exchange, r (blue circles)
and of the unfolding rate,
t
U
(red circles) as a
function of the pulling force. The solid blue line is
a fit which yields a distance to the transition state
for disulfide bond reduction of
L
1
of the protein up to the position of
the disulfide bond. If DTT is present in the
solution, disulfide bond reduction can occur,
allowing for the extension of the trapped residues
D
D
D
x
r
¼
0.34 Å while
the solid red line yields a value of
1.75 Å for
the unfolding distance to the transition state of
I27 [105].
D
x
U
¼
L
2
. (C) Force-clamp experiment showing a
double-pulse protocol which separates the
Figure 13.9C. We found that over a range of 100 to 400 pN of applied force the rate of
disul
de bond reduction was accelerated 10-fold, demonstrating that mechanical
force can indeed catalyze this chemical reaction.
The observed force dependence of the rate of disul
de bond reduction by DTT
was found to be much less sensitive than the rate of I27 unfolding (Figure 13.9D). The
weaker force dependence indicates a much shorter value of
D
xfordisul
de bond
reduction (
0.34 Å in Figure 13.9D) [105]. Indeed, it was remarkable that the
measured distance to the transition state of this S
N
2-type chemical reactionwas in close
agreement with disul
de bond lengthening at the transition state of thiol
-
disul
de
D
x
r
¼
