Related ArticlesNMR exchange broadening arising from specific low affinity protein self-association: analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1.
J Biomol NMR. 1999 Aug;14(4):307-20
Authors: Pfuhl M, Chen HA, Kristensen SM, Driscoll PC
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 microM (pH 6.0, 25 degrees 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 tau 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 beta-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 approximately 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.
[Question from NMRWiki Q&A forum] Line broadening in nmr
Line broadening in nmr
Hello can some body help me in understanding the line broadening effect in NMR? I am working with protein and small molecules and I observed severe broadening at a protein to ligand ratio 1:20. I am pretty sure that its not aggregation since i have the free ligand spectrum without the protein which didnt showed any broadening effect.
Hope somebody will guide me..
Check if somebody has answered this question on NMRWiki QA forum
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NMR exchange broadening arising from specific low affinity protein self-association:
Linear Mode
