Detection of the Ca2+-dependent Association between CaM and M13 Peptide with FCCS
Introduction
Calmodulin (CaM) acts as an intracellular calcium sensor that translates the Ca2+ signal into a variety of cellular processes. Ca(2+)-CaM recognition of a short polypeptide segment in target proteins induces conformational changes in both CaM and the target, enabling the target protein to become functionally active. This time, the Ca2+ dependent association between CaM and M13 peptide(C terminal of Myosin-light-chain kinase) was detected using FCCS.
Substance
The recombinant proteins (His-tag-mAG-CaM, M13-mKeima-His-tag) from E. coli were purified using Ni-NTA agarose.
Measurement
The both recombinant proteins (~10nM, buffer: 150 mM KCl, 50 mM HEPES KOH pH 7.4, 100 uM EGTA) were complexed in a cuvette and measured（1）. CaCl2 was then added to the cuvette to obtain a concentration of 200 uM （2）. Finally, EGTA was added to the cuvette to obtein a concentration of 1 mM （3）.
Result
The amplitude of the cross-correlation was low in the absence of Ca2+（1）, but increased after the addition of Ca2+ because of CaM-M13 binding（2）. Finally, the addition of EGTA to trap the Ca2+ caused debinding of CaM-M13 and the amplitude of the cross-correlation decreased（3）.
