TY - JOUR
T1 - Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein
AU - Siddique, Md Shahjahan P.
AU - Mogami, George
AU - Miyazaki, Takashi
AU - Katayama, Eisaku
AU - Uyeda, Taro Q.P.
AU - Suzuki, Makoto
N1 - Funding Information:
The authors thank Drs. H. Higuchi and N. Sasaki for their support in carrying out the experiments. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (No.16041203, No.17040004) and Grants-in-Aid for Scientific Research (No.16370070, No.17053004) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2005/12/2
Y1 - 2005/12/2
N2 - Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480 nm using 365- nm excitation. The relative increases of fluorescence intensity at 390 nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.
AB - Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480 nm using 365- nm excitation. The relative increases of fluorescence intensity at 390 nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.
KW - Actin filament
KW - Acto-S1 chimera
KW - Cooperative structural change
KW - Dictyostelium
KW - Fluorescence spectroscopy
KW - Intermediate state
KW - Motility function
UR - http://www.scopus.com/inward/record.url?scp=27144435137&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27144435137&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2005.09.159
DO - 10.1016/j.bbrc.2005.09.159
M3 - Article
C2 - 16226711
AN - SCOPUS:27144435137
SN - 0006-291X
VL - 337
SP - 1185
EP - 1191
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -