Biomedical Engineering Reference
In-Depth Information
chemical exchange regime. A similar lack of broadening was observed in the
earlier work,
33
but was not analysed.
To be sure that we were not fooled by the TROSY scheme, we measured
15
N
spin relaxation rates for DnaJ(1-70) in the absence and presence of DnaK. The
relaxation data were first fitted using the spectral density function
34
S
2
t
c
1z(vt
c
)
2
J(v)~
ð
10
:
35
Þ
for both R
1
and R
2
, together with an R
ex
exchange term for R
2
. We used an in-
house grid-search program. For isolated DnaJ(1-70), we obtained a molecular
rotational correlation time of 5.2 ns, with good statistics for the fit and little
R
ex
(see Table 10.4). A fit using eqn (10.35) to the (extrapolated)
15
N
relaxation data for DnaJ 100% bound to the 70 kDa DnaK yielded t
c
5 8.0 ns
(see Table 10.4). This result shows that J-70 moves around quite freely while it
is stoichiometrically and saturably bound to DnaK, given that the rotational
correlation time for DnaK by itself is 28 ns.
25
But the fit is not very good
(Table 10.4) and requires excessive
15
N exchange broadening which, in fact, is
not observed at all. However, the
15
N relaxation data of bound DnaJ(1-70)
can be fitted in a meaningful way with a model in which DnaJ is dynamically
tethered to DnaK in a complex with a 28 ns overall correlation time.
We proceeded and fitted the data for bound DnaJ with the 'model-free'
density function
34
1z(vt
c
)
2
z
(1{S
2
)t
S
2
t
c
ð
10
:
36
Þ
J(v)~
1z(vt)
2
As Table 10.4 shows, this fit returns S
2
5 0.37 and a value of 3.8 ns for t
e
,
which is close to the value of t
c
for free DnaJ(1-70). The statistics of this fit are
also much better. We calculated the order parameter for a variety of overall
correlation times, as shown in Table 10.4. It shows that a fit with the lowest
value for R
ex
and a physically reasonable value for t
e
is indeed obtained
around t
c
5 28 ns. This lends much credence to validity of our assumptions.
Because the J-domain happens to be an a-helical bundle, most NH vectors in
DnaJ(1-70) point in the same direction (parallel to the helical axes). Hence the
average order parameters of these vectors describe the average order parameter
of the DnaJ-domain itself, and model-free can be used to describe the motion
of DnaJ(1-70) with respect to DnaK.
If the motion of DnaJ(1-70) with respect to DnaK can be described as
tethered to one point and moving around in a cone with respect to that point,
one obtains from ref.
34
for S
2
5 0.37
2
cos h 1zcos h
ð
Þ
S
2
~
ð
10
:
37
Þ
2
an angle of 45u for h, the half-opening angle.
