NMR exchange broadening arising from specific low affinity protein self-association: Analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1 |
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Authors: | Mark Pfuhl Ho A. Chen Søren M. Kristensen Paul C. Driscoll |
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Affiliation: | (1) Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, U.K.;(2) Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 København Ø, Denmark |
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Abstract: | Nuclear spin relaxation monitored by heteronuclear NMR provides a useful method to probe the overall and internal molecular motion for biological macromolecules over a variety of time scales. Nitrogen-15 NMR relaxation parameters have been recorded for the N-terminal domain of the rat T-cell antigen CD2 (CD2d1) in a dilution series from 1.20 mM to 40 M (pH 6.0, 25 °C). The data have been analysed within the framework of the model- free formalism of Lipari and Szabo to understand the molecular origin of severely enhanced transverse relaxation rates found for certain residues. These data revealed a strong dependence of the derived molecular correlation time c upon the CD2d1 protein concentration. Moreover, a number of amide NH resonances exhibited exchange broadening and chemical shifts both strongly dependent on protein concentration. These amide groups cluster on the major -sheet surface of CD2d1 that coincides with a major lattice contact in the X-ray structure of the intact ectodomain of rat CD2. The complete set of relaxation data fit well to an equilibrium monomer–dimer exchange model, yielding estimates of exchange rate constants (kON=5000 M-1 s-1; kOFF=7 s-1) and a dissociation constant (KD 3–6 mM) that is consistent with the difficulty in detecting the weak interactions for this molecule by alternative biophysical methods. The self-association of CD2d1 is essentially invariant to changes in buffer composition and ionic strength and the associated relaxation phenomena cannot be explained as a result of neglecting anisotropic rotational diffusion in the analysis. These observations highlight the necessity to consider low affinity protein self-association interactions as a source of residue specific exchange phenomena in NMR spectra of macromolecular biomolecules, before the assignment of more elaborate intramolecular conformational mechanisms. |
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Keywords: | CD2 crystal contacts exchange broadening nuclear relaxation self-association |
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