Structural Characterization of Proteins with an Attached ATCUN Motif by Paramagnetic Relaxation Enhancement NMR Spectroscopy

• Published in: Journal of the American Chemical Society Vol. 123; no. 40; pp. 9843 - 9847
• Main Authors: Donaldson, Logan W, Skrynnikov, Nikolai R, Choy, Wing-Yiu, Muhandiram, D. Ranjith, Sarkar, Bibudhendra, Forman-Kay, Julie D, Kay, Lewis E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.10.2001
• Subjects: Amino Acid Motifs; Binding Sites; Copper - chemistry; Humans; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Thermodynamics; Ubiquitin - chemistry; Ubiquitin - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja011241p

The use of a short, three-residue Cu2+-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu2+ with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu2+ can be converted into distance restraints and used for structure refinement by using a new routine, PMAG, that has been written for the structure calculation program CNS. The utility of the approach is demonstrated with an application to ATCUN-tagged ubiquitin. Excellent agreement between measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.