Title and Abstract
Introduction
Assignment and Structure Determination
Comparison to other CaM/Peptide Complexes
Relaxation Time Measurements and Backbone Dynamics
Literature

Relaxation Time measurements and Backbone Dynamics

¹⁵N Relaxation time studies of CaM revealed a considerable flexibility for the central part of the protein [7] which links the two domains (residues 76-81), allowing it to easily undergo major structural changes upon binding of target peptides. This increased mobility is revealed by an decreased values of the order parameter S² for these residues. ¹⁵ N Relaxation data data were obtained for CaM/C20W (¹⁵N R₁, R₂ and ¹H/¹⁵N NOE) and CaM/C21W (¹H/¹⁵N NOE). Compared to the ones obtained for CaM the absolute values differ due to differences in temperature and viscosity of the samples (Figure 9).

Figure 9:¹⁵N heteronuclear NOE, R₁- and R₂ relaxation rates (600MHz): CaM (308K)[7], CaM/C20W (303K), CaM/C21W (303K) plotted by residue.

Figure 10: Rotational correlation times t_c and order parameters S² of CaM/C20W ( ) and CaM ( ) versus residue number. For CaM/C20W, the overall correlation times were obtained by optimizing the diffusion tensor seperately for N-terminal () and C-terminal ( ) domain using modelfree and are not identical to the average local correlation times calculated for CaM (, )[7]. The residues with decreased S² are all located outside well defined secondary structure elements. Most also show high solvent accesibility ( ), i. e. a H^N temperature shift coefficients < -7 ppb/K, with the exception of A57, N130and K148.

The anomalities for data in of the central linker are still observable for CaM/C20W, however to a smaller extend than in CaM. A comparison of the ¹H/¹⁵N NOE values of CaM/C20W with the ones of CaM/C21W indicates that the behavior of the central helix in CaM/C20W may already approach the one of a globular complex.
For free CaM, two significantly different correlation times t_c were found for the N- and C-terminal domains (7.12 ns and 6.30 ns, respectively), consistent with the slightly smaller size of the C-terminal part of the molecule and indicating independent motion of the two parts of CaM. An analysis of the CaM/C20W data with the program Modelfree [8] results in almost equal values of tc for the two domains of CaM in this complex with the C-terminal domain having a slightly longer tc (10.1 ns) than the N-terminus (9.8 ns). This correlates with the increase in size of the C-terminal half upon binding of C20W even though a more correlated motion of the domains can not be excludet. The values of the order parameters for the backbone NH vectors in CaM/C20W found for the different residues is analogous to the ones found in free CaM, however the decrease for the central linker residues in less prominent. This is consistent with the C20W complex representing a transition halfway between the fully extended structure of free CaM and the globular structure of most CaM/peptide complexes.