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data set that was not being applied as a restraint. A 15% reduction in
Q
value was
obtained with an ensemble size of two, which was statistically significant. In addition,
several residues were found to be undergoing anisotropic motion and can be better
represented by the two-member ensemble (Fig.
5a
)[
52
]. Later a simulated annealing
with an ensemble size of four to eight on protein GB3 had been found to be optimal
[
71
]. The consensus is that other than residues having large amplitude or anisotropic
motions, most structured residues can be represented with a single structure for
monomeric small proteins because the accuracy of NMR structural coordinates is
well within the measurement uncertainty of the RDCs.
Other studies on ensemble minimization reveal a relatively larger conforma-
tional sampling for ubiquitin [
72
]. In another study, an ensemble of around 100
ubiquitin structures was generated using NMR restrained simulated annealing and
molecular dynamics. Interestingly, such an ensemble covers structural heterogene-
ity observed in 40 or so ubiquitin-ligand complex crystal structures (Fig.
5b
)[
70
].
RDC restraints used in such ensemble minimizations were measured in over 30
different alignment media [
73
]. The biological implication is that ligand induced
ubiquitin conformers preexist in its structural ensemble. Further, the ubiquitin N-H
order parameters derived from RDC analysis [
73
-
76
] were overall lower by 0.1-0.2
than that of MF order parameter derived from relaxation data [
77
], indicating an
appreciable amount of microsecond to millisecond motion that was not observed in
spin relaxation measurement. It is nevertheless an interesting and different conclu-
sion from other findings on ubiquitin [
52
] and GB3 [
41
].
Fig. 5 Structure ensemble of Ubiquitin. (a) Two members (shown in
red
and
blue
) of a typical
ensemble from the two-member size calculation. (Reprinted with permission from [
52
].)
(b) Backbone trace of 40 randomly chosen structures from the ensemble. Residues are colored
by the amount of additional (slower than-
c) mobility as compared with the Lipari-Szabo order
parameters. (Reprinted with permission from [
70
])
t
