pH-dependent conformational changes of wheat germ calmodulin.
Fluorescence energy transfer measurements were carried out between landmarks on wheat germ calmodulin to measure the interdomain distance. Tb3+ ions bound at the four Ca2+-binding sites were used as energy donors, and an organic chromophore, [4-(dimethylamino)-phenyl-4'-azophenyl]maleimide, attached to the single cysteine residue at position 27, was used as the acceptor. At pH's near neutrality all bound Tb3+ ions emit luminescence with shortened lifetimes as a result of energy transfer to the acceptor; at pH 5, however, part of the metal emission becomes unquenched. When the protein is subjected to limited digestion with trypsin in the presence of Ca2+, resulting in the formation of two fragments, each corresponding to half of the molecule, the decay of Tb3+ emission is no longer pH sensitive. These results suggest that, like rabbit skeletal troponin C [Wang, C.-L. A., Zhan, Q., Tao, T., & Gergely, J. (1987) J. Biol. Chem. 257, 8372-8375], wheat germ calmodulin exists in a relatively compact conformation at neutral pH's, but becomes more elongated at pH 5.
Wang
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CL
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000032:analysis*; D004735:Energy Transfer; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D011487:Protein Conformation; D013725:Terbium / Q000378:metabolism; D014908:Triticum / Q000032:analysis*; D014357:Trypsin; D004124:p-Dimethylaminoazobenzene
D002147:Calmodulin; D013725:Terbium; D004124:p-Dimethylaminoazobenzene; D014357:Trypsin; D002118:Calcium