Biomedical Engineering Reference
In-Depth Information
It thus appears that DnaJ(1-70) moves around quite freely while it is bound
to DnaK. Next we wondered if we also could find evidence for this dynamical
behavior from the perspective of DnaK.
10.5.2 A View from the DnaK Perspective
Initially, we attempted to obtain the binding site(s) of DnaJ on DnaK by
chemical shift mapping. However, the chemical shift changes are too small to
interpret. Hence, we proceeded with spin labelling. We mutated and spin-
labelled DnaJ with MTSL [(1-oxyl-2,2,5,5-tetramethyl-D
3
-pyrroline-3-methyl)
methanethiosulfonate] to determine the PRE on the
1
H-
15
N TROSY spectrum
of DnaK. MTSL causes quantifiable line broadening for NMR nuclei in the
range 15-25
˚
of the spin-label, while the resonances of nuclei within 15
˚
are
broadened beyond detection.
35
The first choice was to spin-label DnaJ residue M30. M30 is located on helix
II
. M30 can be mutated to Ala without affecting functionality.
36
Figure 10.18
shows the effect of DnaJ(1-70)M30C-MTSL on DnaK in the presence of ADP
and
NRLLLLTG.
A
contiguous
swatch
comprising
DnaK
residues
206
EIDEVDGEKTFEVLAT
221
is
broadened
away
by
DnaJ(1-70)M30C-
Figure 10.18
The average position of DnaJ(1-70) with respect to DnaK(1-605) in
the presence of ADP and NR as obtained from a molecular dynamics
simulation constrained by PRE distance constraints. DnaK NBD is in
yellow, DnaK SBD in cyan, and Dnaj(1-70) in white. Location of
spin-labels as discussed in the text are indicated. DnaJ M30C-
MTSL(blue) affects the HN resonances on DnaK shown in red.
DnaK V210C-MTSL (red), D326C-MTSL (orange) and T417C-
MTSL (orange) affect, to different extent, the resonances of the
residues on DnaJ indicated in blue. D148C-MTSL, R166C-MTSL and
K421C-MTSL (all in green) had no effect. Adapted from ref. 32.
