We have previously demonstrated that residual dipolar couplings could be measured for a paramagnetic form of the protein myoglobin and that these residual dipolar couplings contained useful structrual. information. Subsequently, in an attempt to quantitatively analyze these data, we found that the residual dipolar data did not fit predictions based on a simple rigid model of the protein and concluded that they were also sensitive to an unusually large range of internal motions. It is now clear that, with the use of new media that amplify alignment, observations are not restricted to just paramagnetic proteins, and that a powerful new route to macromolecular structure determination is at hand. Because use for structure determination presumes the absence of internal motion, or at least a systematic means of eliminating the effects of motion, it has become very important to resolve the issue of whether or not substantial degrees of internal motion do exist in proteins. Among the alternative explanations for the deviations from the simple rigid model predictions that we saw in our initial work on myoglobin is an anomaly in the magnetic susceptibility tensor. To make a quantitative comparison with experiment, the susceptibility tensor of myoglobin had to be calculated from known electronic and structural properties of the isolated monomeric molecule. It is, however, possible for molecules to associate in solution in such a way that they do not exhibit the susceptibility tensor of a monomer. To eliminate this possibility we studied degrees of orientation and residual dipolar coupling as a function of dilution. The experiments were not trivial, as sensitivity decreases on dilution and very precise and well controlled measurements are necessary. The dipolar couplings were measured on the 750 MHz spectrometer, and the data showed no effect of dilution. The search for information that can support or refute the influence of internal motion on this important new source of information is continuing.
Showing the most recent 10 out of 12 publications