Direct Investigation of Slow Correlated Dynamics in Proteins via Dipolar Interactions

Abstract

The synchronization of native state motions as they transition between microstates influences catalysis kinetics, mediates allosteric interactions and reduces the conformational entropy of proteins. However, it has proven difficult to describe native microstates because they are usually minimally frustrated and may interconvert on the μs-ms time scale. Direct observation of concerted equilibrium fluctuations would therefore be an important tool for describing protein native states. Here we propose a strategy that relates NMR cross-correlated relaxation (CCR) rates between dipolar interactions to residual dipolar couplings (RDCs) of individual consecutive HN–N and Hα–Cα bonds, which act as a proxy for the peptide planes and the side chains respectively. Using Xplor-NIH ensemble structure calculations restrained with the RDC and CCR data we observe collective motions on time scales slower than nanoseconds in the backbone for GB3. To directly access the correlations from CCR we develop a structure-free data analysis. The resulting dynamic correlation map is consistent with the ensemble-restrained simulations and reveals a complex network. In general we find that the bond motions are on average slightly correlated, and that the local environment dominates many observations. Despite this, some patterns are typical over entire secondary structure elements. In the β-sheet, nearly all bonds are weakly correlated and there is an approximately binary alternation in correlation intensity corresponding to the solvent exposure/shielding alternation of the side chains. For α-helices there is also a weak correlation in the HN-N bonds and the degree of correlation involving Hα-Cα bonds is directly affected by side-chain fluctuations, while loops show complex and non-uniform behavior.

Distance‐independent Cross‐correlated Relaxation and Isotropic Chemical Shift Modulation in Protein Dynamics Studies

“…However, the data confirms previous findings demonstrating that the H N proton often lies outside the idealized peptide plane. The backbone CCR data set mentioned above (H N N/H N N, H α C α /H α C α , and intraresidual and sequential H N N/H α C α ) also agrees better with an ensemble generated from RDCs, J couplings, 15 N relaxation order parameters and crystallographic B‐factors than with single static structures . In addition, we have used this CCR data set to cross‐validate a structural bundle of GB3 constructed by a physical force field minimally biased by RDCs …”

“…Although there have been attempts to use CCR rates as structural restraints,, they are clearly less explored than other NMR restraints. We foresee two developments that would give multiple‐quantum CCR rates novel significance in structure calculation, dynamics studies and molecular dynamics (MD) force field improvement.…”

“…It is clear that for a detailed assessment of the degree of correlation between the motions of the two interaction axes, these motions have to be quantified individually with a probe that is also sensitive to the slow time scale (μs‐ms). Building on the idea to relate CCR rates to residual dipolar couplings (RDCs) of the individual bonds as given in equation 25,, we have recently mapped out slow correlated motion along the backbone among and between consecutive H N −N and H α −C α bonds of the protein GB3 . For that study, we used the highly accurate and precise H N N/H N N, H α C α /H α C α and intraresidual and sequential H N N/H α C α dipolar CCR rates mentioned above .…”

“…Using the high‐accuracy H N N/H N N, H N N/H α C α and H α C α /H α C α dipolar CCR rates mentioned above, we generated structural models of GB3 and obtained a complex H−X bond motion correlation map . To that purpose, we introduced a harmonic Xplor‐NIH CCR restraint: …”

Detection of Correlated Protein Backbone and Side‐Chain Angle Fluctuations

“…One study has been dedicated to side‐chain bond orientation by means of dipolar H α −C α /H β −C β . Building on the idea of extracting fluctuations of the bonds rather than determination of an average angle,– use of CCR rates has been proposed in cross‐validation of structural ensembles or even to restrain structure calculation with a CCR force field …”