A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in 1H-15N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relaxation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys75 through Thr79 located in the 'central helix' of calmodulin, and for the C-terminal residues Ser147 and Lys148. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr110 through Glu114.
Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.
to be 112°. Molecular mechanics calculations yield a COC bond angle of 111.78°, more in agreement with experimental data.49 In-phase ring stretching vibrational frequencies resulting from Raman spectroscopy for cycloalkanes and cyclic aliphatic ethers have been found to be roughly proportional to the bond angle.47,50 A study on the correlation of planarity of rings with the substitution pattern in chlorinated dibenzodioxin was conducted by Chen, using IR data. 44 The infrared vapor phase spectrum for 2,3,7,8-tetrachlorodibenzodioxin was recorded, and its COC bond angle (a) was calculated from IR data by using mass approximations for the terminal atom in a nonlinear XY2 model and by neglect of the valence force field equations of the symmetric stretch-bending term. Details of these calculations, describing the molecular geometries using IR data for 2,3,7,8-tetrachloro-and other chlorinated dibenzo-
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