Overcoming Solvent Saturation-Transfer Artifacts in Protein NMR at Neutral pH. Application of Pulsed Field Gradients in Measurements of 1H- 15N Overhauser Effects

Abstract

Artifacts due to solvent saturation-transfer effects result in incorrect measurements of 1H- 15N heteronuclear NOE (HNOE). These artifacts are commonly observed in aqueous protein solutions at neutral pH. We describe the application of pulsed field gradients (PFGs) together with long recycle delays in overcoming errors in HNOE measurements which arise from H 2O solvent preirradiation and solvent saturation transfer. Even in the absence of explicit solvent irradiation, the HNOE pulse sequence itself results in a nonequilibrium spin-state distribution of solvent nuclei which can then be transferred by chemical exchange into amide-proton sites. This effect can be avoided by using PFGs for suppression of solvent H 2O together with a recycle delay sufficiently long for the magnetization of water to relax back to equilibrium values during the preparation period. These effects were studied in 15N-enriched human type α transforming growth factor at pH 7.1. Comparisons of PFG-HNOE experiments with and without selective H 2O irradiation and with different recycle times provide estimates of the effects of solvent irradiation on HNOE measurements, which are different for different amide nitrogen-15 nuclei. The amplitudes of these artificial HNOE enhancements are roughly correlated with solvent accessibilities of amide sites in the three-dimensional structure of hTGFα.