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Materials and Methods

Sample Preparation. Recombinant Drosophila CaM was prepared with Escherichia coli (strain AR58) harboring the pAS expression vector (Shatzman & Rosenberg, 1985). Uniform 15N labeling at a level of more than 95% was obtained by growing the bacteria in M9 minimal medium with [15N]-NH4C1 (Isotec Inc.) as the sole nitrogen source. Engineered calmodulin lacks the posttranslational N-acetylation and the trimethylation of the e-amino group of K115. The protein was purified as described previously (Ikura et al., 1990b). A 0.4-mL sample was prepared in 95% H20/5% D20 with a 1.5 mM concentration of CaM, 6.1 mM CaCl2, and 100 mM KC1, pH 6.3. Transverse relaxation time measurements made on a 0.7 mM CaM sample yielded an increase in T2 by~ 6±2% relative to the 1.5 mM solution, suggesting that this re-laxation time (and the rotational diffusion rate) does not in-crease by …