Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of (15)N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR.
 

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of (15)N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR.

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of (15)N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR.

J Biomol NMR. 2011 Aug 9;

Authors: Nadaud PS, Sengupta I, Helmus JJ, Jaroniec CP


Magic-angle spinning solid-state NMR measurements of (15)N longitudinal paramagnetic relaxation enhancements (PREs) in (13)C,(15)N-labeled proteins modified with Cu(2+)-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108-8120, 2009). Using the EDTA-Cu(2+) K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular (15)N-Cu(2+) interactions we have determined a nearly complete set of longitudinal (15)N PREs for a series of microcrystalline samples containing ~10, 15 and 25*mol percent of the (13)C,(15)N-labeled EDTA-Cu(2+)-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental (15)N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu(2+) tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu(2+) ions. To probe this issue we performed a Cu(2+) titration study for K28C-EDTA GB1 monitored by 2D (15)N-(1)H solution-state NMR, which revealed that while for Cu(2+):protein molar ratios of