Analysis of slow interdomain motion of macromolecules using NMR relaxationdata

The interpretation of NMR relaxation data for macromolecules possessing slo w interdomain motions is considered. It is shown how the "extended model-fr ee approach" can be used to analyze N-15 backbone relaxation data acquired at three different field strengths for Xenopus Ca2+-ligated calmodulin. Thi s protein is comprised of two domains connected by two rigid helices joined by a flexible segment. It is possible to uniquely determine all "extended model-free" parameters without any a priori assumptions regarding their mag nitudes by simultaneously least-squares fitting the relaxation data measure d at two different magnetic fields. It is found that the two connecting hel ices (and consequently the domains) undergo slow motions relative to the co nformation in which the two helices are parallel. The time scales and ampli tudes of these "wobbling" motions are characterized by effective correlatio n times and squared-order parameters of approximately 3 ns and 0.7, respect ively. These values are consistent with independent estimates indicating th at this procedure provides a useful first-order description of complex inte rnal motions in macromolecules despite neglecting the coupling of overall a nd interdomain motions.