Abstract:
:The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Bentrop D,Bertini I,Cremonini MA,Forsén S,Luchinat C,Malmendal Adoi
10.1021/bi971022+subject
Has Abstractpub_date
1997-09-30 00:00:00pages
11605-18issue
39eissn
0006-2960issn
1520-4995pii
bi971022+journal_volume
36pub_type
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