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Completely deuterated calmodulin (2HCaM) has been prepared by expressing the chicken gene for CaM in Escherichia coli grown in 2H2O on a deuterated medium. The structural and dynamic properties of a 1:1 CaM/melittin (Mel) complex have been investigated by proton NMR. The spectrum of bound Mel is obtained directly from the spectrum of the 2HCaM X Mel complex and is found to resemble strongly the spectrum of the helical species in methanol rather than that of the random coil species in water. The spectrum of bound CaM is obtained indirectly from the difference spectrum between 1HCaM X Mel and 2HCaM X Mel. Many changes are observed between free and bound CaM and they are distributed in both halves of the molecule, indicating that the binding of Mel affects the structure in both parts of the molecule. The rates of exchange of the amide protons of 2HCaM with 2H2O were compared to those of 2HCaM X Mel. The results showed that most, but not all, of the protons exchanged more slowly in the complex; after 40 hr, the residual peaks number 7 in CaM and greater than 20 in the complex. Again, changes in rates in CaM due to binding of Mel occurred in both halves of the molecule. The relative rates of amide proton exchange in CaM and its complex with Mel prove to be a sensitive criterion of differences in conformational stability and/or structure.
Seeholzer et al. (1986) studied this question.