Covalent binding of ATPgammaS to the nucleotide-binding site in S14C-actin.
We have recently reported on the characterization of beta-actin carrying the mutation S14C in one of the phosphate-binding loops. The present paper describes the attachment of the adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) to actin containing this mutation. Treatment of S14C-actin with ATPgammaS blocked further nucleotide exchange and raised the thermal stability of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATPgammaS and cysteine-14 of the mutant actin. The affinity of the derivatized G-actin for DNase I as compared to wild-type ATP-actin was lowered to a similar extent as that of ADP.AlF(4)-actin. The derivatized actin polymerized slower than ATP-actin but faster than ADP-actin. Under these conditions the bound ATPgammaS was hydrolyzed, suggesting the formation of a state corresponding to the transient ADP.P(i)-state. ATPgammaS-actin interacted normally with profilin, whereas the interaction with actin depolymerizing factor (ADF) was disturbed, as judged on the effects of these proteins on actin polymerization.
Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
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Schüler
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Schutt
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Lindberg
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Karlsson
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D003285:Contractile Proteins; D004355:Drug Stability; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051304:Profilins; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000199:Actins; D001704:Biopolymers; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate