Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum

G. M. Strasburg, M. Hogan, W. Birmachu, D. D. Thomas, C. F. Louis

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71 Scopus citations


Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM·TnI·TnT ternary complex. This suggests that both subunits are within 10 Å of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with K(d) = 6 x 10-8 M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.

Original languageEnglish (US)
Pages (from-to)542-548
Number of pages7
JournalJournal of Biological Chemistry
Issue number1
StatePublished - 1988


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