Thursday, June 11, 2009

J. Am. Chem. Soc., 2009, 131 (23), pp 8108–8120

Paramagnetic Ions Enable Tuning of Nuclear Relaxation Rates and Provide Long-Range Structural Restraints in Solid-State NMR of Proteins
Philippe S. Nadaud, Jonathan J. Helmus, Stefanie L. Kall and Christopher P. Jaroniec

Magic-angle-spinning solid-state nuclear magnetic resonance (SSNMR) studies of natively diamagnetic uniformly 13C,15N-enriched proteins, intentionally modified with side chains containing paramagnetic ions, are presented, with the aim of using the concomitant nuclear paramagnetic relaxation enhancements (PREs) as a source of long-range structural information. The paramagnetic ions are incorporated at selected sites in the protein as EDTA−metal complexes by introducing a solvent-exposed cysteine residue using site-directed mutagenesis, followed by modification with a thiol-specific reagent, N-[S-(2-pyridylthio)cysteaminyl]EDTA-metal. Here, this approach is demonstrated for the K28C and T53C mutants of B1 immunoglobulin-binding domain of protein G (GB1), modified with EDTA-Mn2+ and EDTA-Cu2+ side chains. It is shown that incorporation of paramagnetic moieties, exhibiting different relaxation times and spin quantum numbers, facilitates the convenient modulation of longitudinal (R1) and transverse (R2, R1ρ) relaxation rates of the protein 1H, 13C, and 15N nuclei. Specifically, the EDTA-Mn2+ side chain generates large distance-dependent transverse relaxation enhancements, analogous to those observed previously in the presence of nitroxide spin labels, while this phenomenon is significantly attenuated for the Cu2+ center. Both Mn2+ and Cu2+ ions cause considerable longitudinal nuclear PREs. The combination of negligible transverse and substantial longitudinal relaxation enhancements obtained with the EDTA-Cu2+ side chain is especially advantageous, because it enables structural restraints for most sites in the protein to be readily accessed via quantitative, site-resolved measurements of nuclear R1 rate constants by multidimensional SSNMR methods. This is demonstrated here for backbone amide 15N nuclei, using methods based on 2D 15N−13C chemical shift correlation spectroscopy. The measured longitudinal PREs are found to be highly correlated with the proximity of the Cu2+ ion to 15N spins, with significant effects observed for nuclei up to 20 Å away, thereby providing important information about protein structure on length scales that are inaccessible to traditional SSNMR techniques.

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