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.
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 T) 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 (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-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 (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). 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.
Hall
Jennifer B
JB
Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742, USA.
Fushman
David
D
D000588:Amines / Q000737:chemistry; D016880:Anisotropy; D004574:Electromagnetic Fields; D006860:Hydrogen Bonding; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012996:Solutions / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
D000588:Amines; C029152:G-substrate; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D012996:Solutions; D025801:Ubiquitin