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

摘要

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 T) to determine the ~(15)N 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 ~(15)N 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 ~(15)N 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 ~(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 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 ~(15)N CSA for accurate analysis of protein dynamics from ~(15)N relaxation measurements.