title: Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: A J
authors.initials: AJ
authors.lastname: Dingley
authors.forename: J P
authors.initials: JP
authors.lastname: Mackay
authors.forename: G L
authors.initials: GL
authors.lastname: Shaw
authors.forename: B D
authors.initials: BD
authors.lastname: Hambly
authors.forename: G F
authors.initials: GF
authors.lastname: King