Biologically active fluorescent derivatives of spinach calmodulin that report calmodulin target protein binding.
Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing a 4.6-fold fluorescence enhancement. Caldesmon bound with a Kd of 250 nM, causing a 2-fold fluorescence enhancement. Calcineurin (CaN) bound to MIANS-CaM with a Kd less than 5 nM, causing an 80% increase in fluorescence. On the other hand, binding of the CaM antagonist drugs prenylamine and calmidazolium or the potent peptide antagonist melittin did not alter MIANS fluorescence. MIANS-CaM activated brain cGMP phosphodiesterase and CaN as effectively as unlabeled CaM. Spinach CaM was also labeled with three other sulfhydryl reagents, 6-acryloyl-2-(dimethylamino)naphthalene, (2,5-dimethoxy-4-stilbenyl)maleimide, and rhodamine X maleimide. CaN bound to the highly fluorescent rhodamine X maleimidyl-CaM with a Kd of 1.4 nM, causing a 25% increase in polarization. Both MIANS-CaM and rhodamine X-CaM were used to monitor the Ca2+ dependence of the interaction between CaM and CaN. Half-maximal binding occurred at pCa 6.7-6.8 in the absence of Mg2+, or at pCa 6.3 in the presence of 3 mM Mg2+. In both cases, the dependence of the interaction was cooperative with respect to Ca2+ (Hill coefficients of 1.7-2.0). Use of these fluorescent CaMs should allow accurate monitoring of CaM interactions with its target proteins and perhaps their localization within the cell.
Mills
J S
JS
Department of Physiological Chemistry, Ohio State University Medical Center, Columbus 43210.
Walsh
M P
MP
Nemcek
K
K
Johnson
J D
JD
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D013439:Sulfhydryl Reagents; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D009219:Myosin-Light-Chain Kinase; D003545:Cysteine; D002118:Calcium