Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy.
The use of a short, three-residue Cu(2+)-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 Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu(2+) 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.
Donaldson
L W
LW
Department of Medical Genetics, University of Toronto, Toronto, ON, Canada, M5S 1A8.
Skrynnikov
N R
NR
Choy
W Y
WY
Muhandiram
D R
DR
Sarkar
B
B
Forman-Kay
J D
JD
Kay
L E
LE
D020816:Amino Acid Motifs; D001665:Binding Sites; D003300:Copper / Q000737:chemistry*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D025801:Ubiquitin; D003300:Copper