Variability of the 15N Chemical Shielding Tensors in the B3 Domain of Protein G from 15N Relaxation Measurements at Several Fields

摘要

We applied a combination of ¹⁵N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 Tesla) to determine the ¹⁵N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the ¹⁵N chemical shielding tensor, similar to those in ubiquitin. Assuming a Gaussian distribution of the ¹⁵N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02-Å NH-bond length) and the site-so-site CSA variability is ±17.6 to ±21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H or recently, using “meta-analysis” for ubiquitin. Standard interpretation of ¹⁵N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform ¹⁵N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of ¹⁵N CSA for accurate analysis of protein dynamics from ¹⁵N relaxation measurements.