MEASURING MACROMOLECULAR DIFFUSION USING HETERONUCLEAR MULTIPLE-QUANTUM PULSED-FIELD-GRADIENT NMR

We have previously shown that H-1 pulsed-field-gradient (PFG) NMR spec troscopy provides a facile method for monitoring protein self-associat ion and can be used, albeit with some caveats, to measure the apparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol. NMR , 6, 321-328]. In this paper we show that, for N-15-labelled proteins, selection of H-1-N-15 multiple-quantum (MQ) coherences in PFG diffusi on experiments provides several advantages over monitoring H-1 single- quantum (SQ) magnetization. First, the use of a gradient-selected MQ f ilter provides a convenient means of suppressing resonances from both the solvent and unlabelled solutes. Second, H-1-N-15 zero-quantum cohe rence dephases more rapidly than H-1 SQ coherence under the influence of a PFG. This allows the diffusion coefficients of larger proteins to be measured more readily. Alternatively, the gradient length and/or t he diffusion delay may be decreased, thereby reducing signal losses fr om relaxation. In order to extend the size of macromolecules to which these experiments can be applied, we have developed a new MQ PFG diffu sion experiment in which the magnetization is stored as longitudinal t wo-spin order for most of the diffusion period, thus minimizing sensit ivity losses due to transverse relaxation and J-coupling evolution.