Fluorescence probe study of Ca2+-dependent interactions of calmodulin with calmodulin-binding peptides of the ryanodine receptor.
We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca(2+) increase in a physiological range of Ca(2+) (0.1-2 microM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca-CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca(2+), and a large and fast increase at high Ca(2+). Thus, the badan probe attached to CaM Cys(34) can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM-CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM-CaMBP complex are altered by calcium.
Gangopadhyay
Jaya Pal
JP
Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Grabarek
Zenon
Z
Ikemoto
Noriaki
N
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D015081:2-Naphthylamine; D002118:Calcium