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We have previously shown that (1)H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile method for monitoring proteinself-association and can be used, albeit with some caveats, to measure theapparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol.NMR, 6, 321-328]. In this paper we show that, for(15)N-labelled proteins, selection of(1)H-(15)N multiple-quantum (MQ) coherences in PFGdiffusion experiments provides several advantages over monitoring(1)H single-quantum (SQ) magnetization. First, the use of agradient-selected MQ filter provides a convenient means of suppressingresonances from both the solvent and unlabelled solutes. Second,(1)H-(15)N zero-quantum coherence dephases morerapidly than (1)H SQ coherence under the influence of a PFG.This allows the diffusion coefficients of larger proteins to be measuredmore readily. Alternatively, the gradient length and/or the diffusion delaymay be decreased, thereby reducing signal losses from relaxation. In orderto extend the size of macromolecules to which these experiments can beapplied, we have developed a new MQ PFG diffusion experiment in which themagnetization is stored as longitudinal two-spin order for most of thediffusion period, thus minimizing sensitivity losses due to transverserelaxation and J-coupling evolution. |
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