3.3 Molecular dynamics simulation
We sought to characterize the locally disordered intermediate
conformation of OspA from PRE data. However, a conversion ofR 2PRE into distance is not straightforward in
this case since the protein exists as a mixture of different
conformational states, including ~80% locally
disordered, ~20% native, and minimal amounts of
completely unfolded protein. Even for the completely unfolded state, it
is not easy to reconstruct the probability distribution of the distance
between the paramagnetic probe and each amide proton, as multiple
sources of stochastic dynamics in the system must be
considered.30 Here, to produce potential
structural models of the intermediate conformations of WT* OspA, we
performed a 1 µs MD simulation with the NVT ensemble (350 K). The
MD simulation demonstrated that temperature-induced disordering of the
β-sheet began at the C-terminal region, followed by the central region.
Distance distributions were calculated between the paramagnetic probe
and each amide proton for four different time-regions in the MD
trajectory, corresponding to the different levels of disorder in
β9–β11. Figure S14 presents the distance distribution between the
Cβ of residue 118/128/140 (a substitute for the
paramagnetic center) and the amide proton of residue 37 for the folded,
partially disordered, and completely disordered ensembles. A specific
simulation snapshot was obtained in the folded and partially disordered
ensembles, in which β11, β10, and β9 are disordered (Figure 8A). The
distance for the closest approach d 0 and that of
the maximum separation L for the ensembles were obtained from the
distance distribution. In the partially disordered and completely
disordered ensembles, the distance distribution became wider than that
of the folded ensemble.I para/I dia profiles of the
folded and the partially disordered ensembles, as predicted by the MD
simulation, are shown in Figure 8B (see Methods). Increasing the ratios
for residues 33, 93, and 94 of the D118C variant can be explained by the
partially disordered ensemble 3 (PDE3, Figure S8). Meanwhile, the
increased ratios of residues 80–83, 103, and 106 of the E128C may have
resulted from partially disordered ensembles 1 and 2 (PDE1 and PDE2).
Further, PDE1 and PDE2 can explain the incremental ratios for residues
36, 37, 94, and 95 of the A140C variant. However, no ensemble can
explain the decreases observed in the ratios of residues 33-37, 59, and
62. In short, any single specific ensemble cannot explain the entire
profile of I para/I dia.
Moreover, there is no evidence to suggest that the new peaks
corresponding to the residues in the central β-sheet appeared in the
central portion of the spectrum. Hence, we do not deny broadening and
missing of HSQC cross-peaks owing to conformational dynamics; however,
we speculate that the central β-sheet does not become unfolded, but
rather partially disordered, and contains heterogeneous conformations in
the intermediates.
Thus, the intermediate state of the protein is likely a mixture of
conformations, with different levels of partial disorder in the central
β-sheet. This idea is consistent with the conformational heterogeneity
of the equilibrium intermediate mapped by native state hydrogen exchange
NMR and scanning mutagenesis.10,11 To satisfy
the entire profile more quantitatively, PRE should be measured under
conditions in which the intermediate state is more dominantly
stabilized, and larger numbers of snapshots should be generated via MD
simulation of the partially disordered protein.