Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein.
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 480nm using 365-nm excitation. The relative increases of fluorescence intensity at 390nm 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.
Siddique
Md Shahjahan P
MS
Department of Materials Processing, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-02, Sendai 980-8579, Japan.
Mogami
George
G
Miyazaki
Takashi
T
Katayama
Eisaku
E
Uyeda
Taro Q P
TQ
Suzuki
Makoto
M
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence
D000199:Actins; D015879:Myosin Subfragments; D011108:Polymers; D011721:Pyrenes; D011993:Recombinant Fusion Proteins; C030984:pyrene; D000251:Adenosine Triphosphatases; D009218:Myosins