Translational Diffusion of Macromolecular Assemblies Measured Using Transverse-Relaxation-Optimized Pulsed Field Gradient NMR

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 09/2011; 133(41):16354-7. DOI: 10.1021/ja206531c
Source: PubMed


In structural biology, pulsed field gradient (PFG) NMR spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for NMR structure determination or for crystallization trials. This paper describes two transverse-relaxation-optimized (TRO) (15)N-filtered PFG stimulated-echo (STE) experiments for studies of macromolecular translational diffusion in solution, (1)H-TRO-STE and (15)N-TRO-STE, which include CRINEPT and TROSY elements. Measurements with mixed micelles of the Escherichia coli outer membrane protein X (OmpX) and the detergent Fos-10 were used for a systematic comparison of (1)H-TRO-STE and (15)N-TRO-STE with conventional (15)N-filtered STE experimental schemes. The results provide an extended platform for evaluating the NMR experiments available for diffusion measurements in structural biology projects involving molecular particles with different size ranges. An initial application of the (15)N-TRO-STE experiment with very long diffusion delays showed that the tedradecamer structure of the 800 kDa Thermus thermophilus chaperonin GroEL is preserved in aqueous solution over the temperature range 25-60 °C.

Download full-text


Available from: Kurt Wüthrich, Feb 17, 2015
  • Source
    • "Unlike the methods described above, this technique can be performed without the introduction of labels, and in ideal cases can distinguish individual components of the membrane, making it a valuable analytical method. The membranes to be studied need to be aligned with respect to the experimental frame of reference, which may be achieved using bilayers formed on glass plates, or by using magnetically aligned bicelles (Horst et al. 2011, Macdonald and Soong 2011). During the signal acquisition sequence, two pulsed magnetic fields are applied at different times that lead to changes of intensity for molecules that have moved between the pulses, with the magnitude of the change dependent on the extent to which molecules have moved, i.e., D L . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.
    Full-text · Article · May 2012 · Molecular Membrane Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Aqueous solutions of the detergent Fos-10 (n-decylphosphocholine) without and with addition of the integral membrane protein (IMP) OmpX (outer membrane protein X) have been characterized using pulsed field gradient-stimulated echo (PFG-STE) NMR experiments for measurements of translational diffusion coefficients. Effective diffusion coefficients for Fos-10 micelles in the absence of OmpX were obtained by observation of NMR signals from 10-bromodecan-1-ol that had been inserted into the micelles, and in the presence of OmpX by NMR observation of the protein. It is thus shown that solutions of Fos-10-reconstituted OmpX can be quantitatively described as a mixture of Fos-10 monomers, uniform Fos-10 micelles, and uniform OmpX-containing Fos-10 micelles, with Fos-10 monomers in fast exchange between the pools of these three species. This result establishes an avenue for efficient determination of the effective translational diffusion coefficients of IMP-containing detergent micelles based on observation of the intense detergent NMR signals, which is also applicable with unlabeled IMPs. This monitoring of the species present in a given IMP solution contributes to improved guidelines for rational selection of detergent and buffer conditions in structural studies of integral membrane proteins.
    No preview · Article · Feb 2012 · The Journal of Physical Chemistry B
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel NMR approach allows one to efficiently determine translational diffusion coefficients of macromolecules in solution. This method for Signal Optimization with Recovery in Diffusion Delays (SORDID) monitors transport occurring during the recovery times between consecutive scans so that the duration of the measurements can be reduced approximately by a factor two.
    Preview · Article · Apr 2012 · Chemical Communications
Show more