Solution NMR Measurement of Amide Proton Chemical Shift Anisotropy in 15N-Enriched Proteins. Correlation with Hydrogen Bond Length§

Cross-correlation between 15N-1H dipolar interactions and 1HN chemical shift anisotropy (CSA) gives rise to different relaxation rates of the doublet components of 1H-{15N} peptide backbone amides. Two schemes for quantitative measurement of this effect are described and demonstrated for samples of uniformly 15N-enriched ubiquitin and perdeuterated 15N-enriched HIV-1 protease. The degree of relaxation interference correlates with the isotropic 1HN chemical shift, and results indicate that an upfield change of the most shielded principal components of the CSA tensor is correlated with an approximately 2-fold larger downfield shift of the average of the other two components. The magnitude of the relaxation interference is large in â-sheet and considerably smaller in R-helices. This correlation is not dominated by the backbone geometry but reflects the slightly longer hydrogen bond length in helices compared to â-sheet. The smallest relaxation interference effect in ubiquitin is observed for Ser20-HN and Ile36-HN, which are the only two amide protons that are not hydrogen bonded in the crystal structure of ubiquitin, inaccessible to solvent, and not highly mobile. There has been extensive interest in developing a quantitative understanding of the relation between protein structure and chemical shifts.1-8 Most work so far has focused on 13C and 15N, and empirical correlations between protein structure and isotropic 13CR and 13Câ chemical shifts have been confirmed by ab initio calculations.3,9 HR chemical shifts empirically were also found to be quite sensitive to secondary structure.6-8,10-13 As was the case for 13CR shifts, this dependence primarily results from the chemical shift dependence on the φ and ψ backbone torsion angles6 and not from hydrogen bonding itself. In contrast, it is well established that the isotropic amide proton chemical shift is strongly influenced by hydrogen bonding.13-16 Measurement of the relatively small 1H chemical shift anisotropy (CSA) in the solid state is technically challenging, and only a very modest library of 1H CSA values has been accumulated.18 For protons involved in a hydrogen bond, these data indicate that the downfield change in isotropic shift associated with increasing hydrogen bond strength is accompanied by an increase in 1H CSA.18 In peptidic systems, only three studies on amide proton (HN) CSA have been published: Reimer and Vaughan reported data for acetanalide,19 using multipulse 1H NMR. Opella and coworkers reported CSA values from single crystal 2H NMR of N-acetyl-D,L-valine,20 and, more recently, this group used a three-dimensional solid state NMR experiment to obtain the LeuHN shielding tensor in Ala-[15N]Leu.21 These data indicate that, although the HN CSA tensor can deviate from axial symmetry, its most shielded tensor component (σ33) is roughly collinear with the N-H bond. In this study, we show that quantitative information on the CSA tensor of individual amide protons in 15N-enriched proteins can be readily obtained from measurement of relaxation interference between the 1HN CSA and the 1HN-15N dipolar relaxation mechanisms. Relaxation of peptide 1HN spins in 15Nenriched proteins is dominated by 1H-1H dipolar interactions, 1H-15N dipolar coupling, and 1HN CSA. Assuming, for convenience, that the 1HN CSA tensor is axially symmetric, with * Corresponding author: Ad Bax, Building 5, Room 126, NIH, Bethesda, MD 20892-0520. † Laboratory of Biophysical Chemistry. ‡ Laboratory of Chemical Physics. §