caa a22 4500 445325267 CHVBK 20180317142718.0 cr unu---uuuuu 170323e20111101xx s 000 0 eng 10.1007/s10858-011-9536-y doi (NATIONALLICENCE)springer-10.1007/s10858-011-9536-y Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of 15N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR [Elektronische Daten] [Philippe Nadaud, Ishita Sengupta, Jonathan Helmus, Christopher Jaroniec] Magic-angle spinning solid-state NMR measurements of 15N longitudinal paramagnetic relaxation enhancements (PREs) in 13C,15N-labeled proteins modified with Cu2+-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-Cu2+ 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 15N-Cu2+ interactions we have determined a nearly complete set of longitudinal 15N PREs for a series of microcrystalline samples containing ~10, 15 and 25mol percent of the 13C,15N-labeled EDTA-Cu2+-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 15N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu2+ 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 Cu2+ ions. To probe this issue we performed a Cu2+ titration study for K28C-EDTA GB1 monitored by 2D 15N-1H solution-state NMR, which revealed that while for Cu2+:protein molar ratios of ≤ 1.0 Cu2+ binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu2+ ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu2+ binding sites, and indeed many of these residues were ones found to display the elevated longitudinal 15N PREs in the solid phase. Springer Science+Business Media B.V., 2011 Solid-state NMR nationallicence Magic-angle spinning nationallicence Paramagnetic relaxation enhancement nationallicence Pseudocontact shift nationallicence Spin label nationallicence Metal ion nationallicence Protein structure nationallicence Nadaud Philippe Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut Sengupta Ishita Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut Helmus Jonathan Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut Jaroniec Christopher Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut Journal of Biomolecular NMR Springer Netherlands 51/3(2011-11-01), 293-302 0925-2738 51:3<293 2011 51 10858 https://doi.org/10.1007/s10858-011-9536-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9536-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nadaud Philippe Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut NATIONALLICENCE 700 1- Sengupta Ishita Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut NATIONALLICENCE 700 1- Helmus Jonathan Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut NATIONALLICENCE 700 1- Jaroniec Christopher Department of Chemistry, The Ohio State University, 100 West 18th Avenue, 43210, Columbus, OH, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/3(2011-11-01), 293-302 0925-2738 51:3<293 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer