An overall increase in 15N transverse relaxation rate values, R2, is observed upon titration of Bcl-xL with cytochrome c, which is consistent with an increase in the overall tumbling correlation time upon complex formation [18].
In the most common approach to the analysis of protein dynamics, relaxation data – in the form of longitudinal relaxation rates (R1), transverse relaxation rates (R2), and heteronuclear NOEs (Nuclear Overhauser Effects) – are used to derive microdynamic parameters that describe both the overall tumbling and the internal motions of a macromolecule.
The internal dynamics and overall tumbling were fit with the program FAST-ModelFree.
This result indicates that the overall tumbling of the peptide backbone has lowered.
Therefore, tryptophan anisotropy is typically sensitive to side-chain rotation, but the overall tumbling of MscS is too slow.
With the domain-specific overall tumbling times set, we were able to fit the site-specific side-chain internal motion parameters, SAXIS2 and τe.
For the overall tumbling correlation time τm, we used the Levenberg Marquardt algorithm and fit the ratios R2(N / R1(N) of each domain (WW and PPIase) to get domain-specific τm values.
The parameter τ satisfies 1/τ = 1/τm + 1/τe, where τm is the global correlation time for overall tumbling, and τe is a site-specific correlation time related to motions underlying SAXIS2.
In the case of the NOESY experiment, inter-proton distances (rij) were averaged over the ensemble according to 〈 rij−6〉 and the resultant matrix converted to a relaxation rate matrix by multiplication by spectral densities estimated from an overall tumbling correlation time (τc).
It is noteworthy that even though the self-association also increases the overall tumbling of the N-terminal part of Cby, this segment of the protein is highly flexible, and a large part of it does not interact with the C-terminal region.
