ROTATIONAL-DYNAMICS OF CALCIUM-FREE CALMODULIN STUDIED BY N-15-NMR RELAXATION MEASUREMENTS

The backbone motions of calcium-free Xenopus calmodulin have been char acterized by measurements of the N-15 longitudinal relaxation times (T -1) at 51 and 61 MHz, and by conducting transverse relaxation (T-2), s pin-locked transverse relaxation (T-1 rho), and N-15-{H-1} heteronucle ar NOE measurements at 61 MHz N-15 frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is m ore stable than calmodulin's C-terminal half, slowly exchanging backbo ne amide protons are found in all eight alpha-helices and in three of the four short beta-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a 'molten gl obule' type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 mu s, which affects many of the C-terminal domain residues. This results in significant shortening of the N-15 T-2 values relative to T-1 rho, whereas the T-1 rho and T-2 values are of similar magnitude in the N- terminal half of the protein. A model in which the motion of the prote in is assumed to be isotropic suggests a rotational correlation time f or the protein of about 8 ns but quantitatively does not agree with th e magnetic field dependence of the T-1 values and does not explain the different T, values found for different alpha-helices in the N-termin al domain. These latter parameters are compatible with a flexible dumb bell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30 degrees and a time constant of a bout 3 ns, whereas the overall rotation of the protein occurs on a muc h slower time scale of about 12 ns. The difference in the transverse r elaxation rates observed between the amides in helices C and D suggest s that the change in interhelical angle upon calcium binding is less t han predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. and James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1-17] .