Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 51, Issue 3, Pages 293-302Publisher
SPRINGER
DOI: 10.1007/s10858-011-9536-y
Keywords
Solid-state NMR; Magic-angle spinning; Paramagnetic relaxation enhancement; Pseudocontact shift; Spin label; Metal ion; Protein structure
Categories
Funding
- National Science Foundation [MCB-0745754]
- Eli Lilly and Company
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0745754] Funding Source: National Science Foundation
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Magic-angle spinning solid-state NMR measurements of N-15 longitudinal paramagnetic relaxation enhancements (PREs) in C-13,N-15-labeled proteins modified with Cu2+-chelating tags can yield multiple long-range electron-nucleus distance restraints up to similar to 20 (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 N-15-Cu2+ interactions we have determined a nearly complete set of longitudinal N-15 PREs for a series of microcrystalline samples containing similar to 10, 15 and 25 mol percent of the C-13,N-15-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 N-15 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 N-15-H-1 solution-state NMR, which revealed that while for Cu2+:protein molar ratios of a parts per thousand currency sign 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 N-15 PREs in the solid phase.
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