Structural implications of the chemical modification of Cys(10) on actin.
Cys(10) is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys(10) was modified with fluorescence probes N-(iodoacetyl)N'-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of, J. Biol. Chem. 266:5508-5513). The specificity of Cys(10) modification was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48-375), which contains all of the actin thiols but Cys(10), is not fluorescent. Cys(10) modification exposed a new site on actin to subtilisin cleavage. Edman degradation revealed this site to be between Ala(19) and Gly(20). The modification slightly increased the rate of epsilonATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys(10)-modified F-actin showed small probe-dependent changes in the values of V(max) and K(M). The sliding speed of actin filaments in the in vitro motility assay remained unchanged upon modification of Cys(10). These results indicate that although the labeling of Cys(10) perturbs the structure of subdomain 1, the modified actin remains fully functional. The binding of S1 to actin filaments decreases the accessibility of Cys(10) probes to acrylamide and nitromethane quenchers. Because Cys(10) does not participate directly in either actin polymerization or S1 binding, our results indicate that actin-actin and actin-myosin interactions induce dynamic, allosteric changes in actin structure.
Eli-Berchoer
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L
Department of Oral Biology, Hadassah School of Dental Medicine, Hebrew University, Jerusalem 91120, Israel.
Reisler
E
E
Muhlrad
A
A
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D009282:Naphthalenesulfonates; D017433:Protein Structure, Secondary; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D009218:Myosins; D003545:Cysteine