title: ADP-ribosylation of cross-linked actin generates barbed-end polymerization-deficient F-actin oligomers.
abstract: Actin filament subunit interfaces are required for the proper interaction between filamentous actin (F-actin) and actin binding proteins (ABPs). The production of small F-actin complexes mimicking such interfaces would be a significant advance toward understanding the atomic interactions between F-actin and its many binding partners. We produced actin lateral dimers and trimers derived from F-actin and rendered polymerization-deficient by ADP-ribosylation of Arg-177. The degree of modification resulted in a moderate reduction in thermal stability. Calculated hydrodynamic radii were comparable to theoretical values derived from recent models of F-actin. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar those of wild-type actin, suggesting native or near-native interfaces on the oligomers. Changes in DNase I binding affinity under low and high ionic strength suggested a high degree of conformational flexibility in the dimer and trimer. Polymer nucleation activity was lost upon ADP-ribosylation and rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin, suggesting that interactions with actin binding proteins can overcome the inhibiting activities of ADP-ribosylation. The combined strategy of chemical cross-linking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds.
authors.affiliation: University of Guelph, Guelph, Ontario, Canada.
authors.forename: Alexandru A
authors.initials: AA
authors.lastname: Perieteanu
authors.forename: Danielle D
authors.initials: DD
authors.lastname: Visschedyk
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
authors.forename: John F
authors.initials: JF
authors.lastname: Dawson
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002645:Chickens; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary
chemical_list: D000199:Actins; D018260:Gelsolin; D000246:Adenosine Diphosphate Ribose; D003850:Deoxyribonuclease I