Temperature Dependence of ¹H_N–¹H_N Distances in Ubiquitin As Studied by Exact Measurements of NOEs

Abstract

Although NMR relaxation phenomena provide a great deal of insight into local molecular dynamics, the dynamic picture of biomacromolecules is still largely incomplete, as no method is available to detect motions between atoms that are far apart in the sequence. Our recent investigations (Vögeli et al. J. Am. Chem. Soc. 2009, 131 (47), 17215−17225) indicate that extraction of exact effective distances from NOE rates might allow the determination of such motions. Using this approach, we measured exact effective distances between amide protons in (15)N,(13)C,(2)H-labeled ubiquitin at three temperatures (284, 307, and 326 K). Comparisons among the three data sets reveal that, whereas the correlation-time-corrected cross-relaxation rates increase by 18% from 284 to 307 K, those at 326 K increase by 32% as compared to those at 284 K. Because theoretical considerations indicate that the NOE is largely insensitive to fast motion, as long as the local order parameter (e.g., S(NH)(2)) is larger than 0.5, the effective distance can be calculated from the NOE using its [linear span]r(-6)[linear span] dependency. Doing so, the average NOE increases translate into effective distance changes of 2.4% and 4.0% in the temperature ranges measured. The data presented demonstrate that the determination of quantitative NOEs is a powerful tool for extracting small structural and dynamical changes in a biomolecule.

The nuclear Overhauser effect from a quantitative perspective

“…Very similar conclusions were drawn from the calculation of fast motion order parameters from the RDC-derived EROS ensemble 2k39 [167]. Conclusively, the impact of fast motion can be approximately neglected for H-H spin pairs as long as the local H-X order parameters are larger than 0.5 [165], which is usually fulfilled in globular proteins except for highly flexible tails and loops. …”

“…eNOE-derived motions appear to have smaller amplitudes and are possibly of a different character than present in a 20 ns molecular dynamic simulation of ubiquitin in water using the GROMOS force field [33]. Furthermore, a recently published ensemble of structures representing the conformational distribution over time scales up to milliseconds (pdb: 2K39) satisfies the NOEs obtained at 307 and 326 K better than the single X-ray structure [165]. However, NOEs at 284 K show a better correlation with the Xray structure which may be caused by the fact that the NMR restraints for the bundle were measured at 298 K [165,167].…”

“…Furthermore, a recently published ensemble of structures representing the conformational distribution over time scales up to milliseconds (pdb: 2K39) satisfies the NOEs obtained at 307 and 326 K better than the single X-ray structure [165]. However, NOEs at 284 K show a better correlation with the Xray structure which may be caused by the fact that the NMR restraints for the bundle were measured at 298 K [165,167]. Subsequently, Butts et al showed the method yields very accurate distances (within a few percent of the true values) for rigid organic molecules as examplified on the model compound strychnine [172].…”

“…If motion is not explicitly accounted for in the analysis of The amide proton-amide proton NOE order parameters due to fast motion have been estimated for ubiquitin [165]. The calculation is based on the experimentally determined H N -N order parameters obtained from relaxation measurements [166].…”

“…Thus, the eNOE is a very attractive tool for the detection of (translational) motions between atoms that are far apart in the sequence. We measured NOE buildups between amide protons in 15 N, 13 C, 2 H-labeled ubiquitin at three temperatures (284, 307 and 326 K, Figure 17) [165]. The temperature changes cause strong alternation of the intensities because the cross-relaxation rates are approximately proportional to the overall tumbling time [3].…”