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.