Rotational Dynamics of Calcium‐Free Calmodulin Studied by 15 N‐NMR Relaxation Measurements

• Published in: European journal of biochemistry Vol. 230; no. 3; pp. 1014 - 1024
• Main Authors: Tjandra, Nico, Kuboniwa, Hitoshi, Ren, Hao, Bax, Ad
• Format: Journal Article
• Language: English
• Published: 01-06-1995
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1995.1014g.x

The backbone motions of calcium‐free
Xenopus
calmodulin have been characterized by measurements of the
15
N longitudinal relaxation times (
T
1
) at 51 and 61 MHz, and by conducting transverse relaxation (
T
2
), spin‐locked transverse relaxation (
T
1ρ
), and
15
N‐{
1
H} heteronuclear NOE measurements at 61 MHz
15
N frequency. Although backbone amide hydrogen exchange experiments indicate that the N‐terminal domain is more stable than calmodulin's C‐terminal half, slowly exchanging backbone amide protons are found in all eight α‐helices and in three of the four short β‐strands. This confirms that the calcium‐free form consists of stable secondary structure and does not adopt a ‘molten globule’ type of structure. However, the C‐terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 μs, which affects many of the C‐terminal domain residues. This results in significant shortening of the
15
N
T
2
values relative to
T
1ρ
, whereas the
T
1ρ
and
T
2
values are of similar magnitude in the N‐terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the
T
1
values and does not explain the different
T
2
values found for different α‐helices in the N‐terminal domain. These latter parameters are compatible with a flexible dumbbell model in which each of calmodulin's two domains freely diffuse in a cone with a semi‐angle of about 30° and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988)
Proteins Struct. Funct. Genet. 3
, 1–17].