Variability of the ¹⁵N Chemical Shielding Tensors in the B3 Domain of Protein G from ¹⁵N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters

Abstract

We applied a combination of 15 N 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 15 N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various modelindependent 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 15 N chemical shielding tensor, similar to those in ubiquitin. Assuming a Gaussian distribution of the 15 N 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 "metaanalysis" for ubiquitin. Standard interpretation of 15 N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15 N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the sitespecific 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 15 N CSA for accurate analysis of protein dynamics from 15 N relaxation measurements.

Density functional calculations of 15N chemical shifts in solvated dipeptides

“…A good agreement with the experimental data is also found for the angle β between the least shielded component (σ 11 ) of the 15 N shielding tensor and the NH bond. The values of β obtained here, from 13.5° to 19.8°, are well in the range of the experimental values (12°-24°) obtained by different NMR techniques, both solution and solid-state Oas et al 1987;Hiyama et al 1988;Shoji et al 1989;Mai et al 1993;Fushman et al 1998;Cornilescu and Bax 2000;Kurita et al 2003;Loth et al 2005;Hall and Fushman 2006;Vasos et al 2006). There seems to be a weak correlation between the β angle and secondary structure, with slightly smaller angles for the β-sheet than for the α-helix (mean β angles are 14.8° and 16.5°, respectively).…”

“…This range, however, is smaller than the 15 N CSA dispersion observed by solution NMR in ubiquitin and GB3 (Fushman et al 1998;Kover and Batta 2001;Hall and Fushman 2006) and by solid-state NMR in GB1 (Wylie et al 2006;Wylie and Rienstra 2008). This likely reflects the fact that these calculations do not take into account the complexity of local electronic environment in proteins, including interactions with neighboring atoms (e.g.…”

Protein conformational flexibility prediction using machine learning

“…Experimental generalized order parameters have been reported for B3 domain of staphyloccocal protein G (GB3) [34], the villin headpiece domain (HP67) [35], the holo frenolicin acyl carrier protein (fren ACP) [36], and Escherichia coli ribonuclease H (RNaseH) [37][38][39][40]. Generalized order parameters were predicted using the structural coordinates from PDB files 1IGD, 1QQV, 1OR5, and 2RN2, respectively.…”

Determination of the antisymmetric part of the chemical shift anisotropy tensor via spin relaxation in nuclear magnetic resonance

“…If amide backbone nitrogen nuclei in slowly tumbling proteins have similar anti-CSA components ͑which remains to be shown͒, it would be important to take these components into consideration whenever the precision of the measurements is better than 1% at 950 MHz. This level of precision can indeed be achieved 24,41,42 but hardly in routine experiments.…”