title: Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.
abstract: The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.
authors.affiliation: Department of Anatomy and Cell Biology, Ruhr-University, Bochum, Germany.
authors.forename: Edda
authors.initials: E
authors.lastname: Ballweber
authors.forename: Ewald
authors.initials: E
authors.lastname: Hannappel
authors.forename: Thomas
authors.initials: T
authors.lastname: Huff
authors.forename: Harald
authors.initials: H
authors.lastname: Stephan
authors.forename: Markus
authors.initials: M
authors.lastname: Haener
authors.forename: Nicole
authors.initials: N
authors.lastname: Taschner
authors.forename: Daniel
authors.initials: D
authors.lastname: Stoffler
authors.forename: Ueli
authors.initials: U
authors.lastname: Aebi
authors.forename: Hans Georg
authors.initials: HG
authors.lastname: Mannherz
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D017348:Microscopy, Electron, Scanning Transmission; D008958:Models, Molecular; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry; D010590:Phalloidine / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011817:Rabbits; D013947:Thymosin / Q000737:chemistry* / Q000378:metabolism*; D000099201:Thymosin beta(4)
chemical_list: D000199:Actins; D001704:Biopolymers; D003432:Cross-Linking Reagents; D018260:Gelsolin; D010590:Phalloidine; D021122:Protein Subunits; D013947:Thymosin; D000099201:Thymosin beta(4)