Assignment and Structure Calculation

Completely 13C/15N labled calmodulin was investigated by a number of triple resonance NMR experiments (CBCA(CO)NH, CBCANH, HCC(CO)NH, HCCH-TOCSY, 13C/15N-NOESY-HSQC). A 12C/14N-filtered NOESY spectrum was recorded to assign the unlabeled peptide C20W and a 12C/14C[f1]-filtered NOESY-HMQC to detect the NOE contacts between calmodulin and the peptide (Figure3 and 4).


Figure 3: Intermolecular NOE's between CaM and Binding Peptides	Figure 4: 2D projection of the ¹²C/¹⁴N[f1]-filtered NOESY-HMQC Spectrum

The present structure calculations are based on 1645 intramolecular distance restraints for CaM, 163 restraints for the peptide C20W and 48 intermolecular NOE contacts observed between the peptide C20W and the C-terminal domain of CaM. These data were supplemented by 61 hydrogen bonds identified by the temperature coefficient of the amide proton chemical shift and 24 distance restraints between the four Ca2+ atoms and protein groups derived from the crystal structure of Ca2+-CaM. Structure calculation was performed with the program XPLOR [4] using a simulated annealing protocol and a subset of 200 structures was generated. The 20 structures of lowest energy were superimposed seperately for the two halves of CaM. Rrmsd values of 0.88 and 1.32 calculated for the bacckbone and all heavy atoms, respectively, were found for the N-terminal half (residues 4-74). For the C-terminal half (residues 85-145 of CaM and 4-18 of C20W) values of 0.53 and 1.04 were obtained.

Figure 5:Ribbon representation showing the C-terminal half of CaM/C20W. The binding of the peptide is stabilized by aromatic contacts between Trp8 and Phe92 as well as side chain interactions between e.g. Ile15 and Val91.