Figure 6: Secondary chemical shift comparison between experiment
and simulation . A comparison between the average secondary chemical
shifts of D0 predicted by the MD simulation (red
line) and those extrapolated from the experiment to zero denaturant
(blue line).
The average radius of gyration, Rg , calculated
from the simulated conformations, was 2.19 ± 0.48 nm. The correspondingRh can be estimated64 to be
2.45 ± 0.61 nm. This is equal, within the error bars, to the
hydrodynamic radius 2.51 ± 0.19 nm obtained as extrapolation to zero
denaturant from the data of Table 1. Another, more qualitative
comparison, was done between the experimental and simulated relaxation
parameters R1 and R2 . The
reason why a direct comparison cannot be done is that a replica-exchange
simulation is efficient in sampling the equilibrium conformations of the
protein at the price of generating an unphysical time-dependent
trajectory, that would be necessary for calculating the NMR relaxation
parameters.
To give an approximate estimate of R1 from the
simulation, we performed 20 plain-MD simulations at fixed temperature
(300K) starting from 20 conformations extracted from the
replica-exchange trajectory. Each simulation lasted for 1 ns, that is
the time scale described by the R1 parameter.
From each simulation we calculated the root mean square fluctuations
(RMSF) around the average conformation. We expected thatR1 is anticorrelated with the RMSF. The
comparison between the experimental R1 and the
(rescaled and shifted) RMSF is displayed in Fig. 7. Although the linear
correlation is not high (r=0.21), 74% of points stay on the same side
with respect to the median (p-value=10-8), suggesting
the two curves indicate similar regions of rigid and flexible residues
(black bars above the curves).
The values of R2 that reflect the conformational
freedom of residues on the µs-ms timescale, were compared with the total
solvent-accessible surface area (SASA) of each amino acid, calculated on
the replica-exchange simulation. Again, the linear correlation is low
(r=0.16) but 68% of the points stay on the same side with respect to
the median (p-value=10-4), indicating that residues
that are experimentally more flexible are those less constrained by
other parts of the polymer in the simulation.