orals 5th Asia-Pacific NMR Symposium 2013

Subtle and not-so-subtle lessons from residual dipolar couplings (#1)

Ad Bax 1 , Alexander S Maltsev 1 , Julien Roche 1 , Alex Grishaev 1
  1. NIH, Bethesda, MD, United States

The antibiotic squalamine, originally isolated from dogfish shark, forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C), pH (4-8), and pressure (1-2500 bar).  Squalamine is positively charged, and comparison of the alignment of ubiquitin in this medium relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide.  High precision residual dipolar couplings (RDCs) measured for the backbone 1H-15N, 15N-13C', and 13C'-13Ca one-bond interactions in squalamine medium fit well to the static structural model previously derived from NMR data.  Inclusion of the new RDCs into the structure refinement procedure resulted in improved agreement between alignment-induced changes in 13C' chemical shift and experimental values, thereby validating the high quality of the static structural model.  Our result indicates that fitting of a single model to experimental data can provide a better description of the time- or ensemble-averaged conformation than do ensemble representations, whereas the latter can capture dynamic aspects of a protein, thus making them valuable complements to one another.  Measurement of RDCs as a function of pressure permits probing of the very small structural changes that immediately precede unfolding, thereby providing detailed insights into the unfolding pathway.