Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength

Hidetake Miyata, Kazuhiko Kinosita, Gerard Marriott

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

F-Actin treated with a half molar equivalent of the heterobifunctional cross-linker, 4-bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE), produced a population of actin oligomers containing 2-14 or more protomers and a significant amount of uncrosslinked actin protomers. The crosslinking reaction is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an adjacent protomer on the genetic helix of F-actin, in a similar; manner to N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers were found to form filaments that sediment after high-speed centrifugation, even in a buffer of low ionic strength [G-buffer: 2 mM tris-hydroxymethylaminomethane (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.3 mM NaN3, 5 mM 2-mercaptoethanol] where affinity between actin protomers usually becomes weak, leading to the depolymerization of native F-actin. By performing the crosslinking reaction in the presence of an environment-sensitive fluorescent label, 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which competes with BNBA-SE for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were obtained which were also stable in G-buffer. Fluorometric analysis of actin labeled with acrylodan (prodan-actin) in these depolymerization-resistant filaments suggested that the molecular environment around Cys 374 of actin protomers is similar to that of actin protomers in native F-actin in F-buffer (G-buffer plus 0.1 M KCl and 1 mM MgCl2). When G-actin labeled with acrylodan was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled actin was incorporated into filaments that were resistant to depolymerization in G-buffer. The emission spectrum of the prodan-actin in these filaments measured in G-buffer was almost identical to that of prodan-actin in native F-actin in F-buffer. Our interpretation of this result is that actin protomers are locked into an F-actin-like conformation in the depolymerization-resistant filament by the subunit interactions they make with crosslinked oligomers. We also present evidence which suggests that the depolymerization rate in G-buffer of filaments made with crosslinked oligomers is much slower than that of native actin because the ends of the depolymerization-resistant filaments are capped with crosslinked oligomers.

Original languageEnglish
Pages (from-to)527-533
Number of pages7
JournalJournal of Biochemistry
Volume121
Issue number3
Publication statusPublished - 1997
Externally publishedYes

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Protein Subunits
Ionic strength
Oligomers
Osmolar Concentration
Actins
Association reactions
Buffers
Depolymerization
Crosslinking
3-nitrobenzoic acid
Actin Cytoskeleton
Esters
Fluorometry
Sodium Azide
Magnesium Chloride

Keywords

  • Acrylodan
  • Actin-actin interface
  • Cooperativity
  • Crosslinking

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength. / Miyata, Hidetake; Kinosita, Kazuhiko; Marriott, Gerard.

In: Journal of Biochemistry, Vol. 121, No. 3, 1997, p. 527-533.

Research output: Contribution to journalArticle

Miyata, Hidetake ; Kinosita, Kazuhiko ; Marriott, Gerard. / Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength. In: Journal of Biochemistry. 1997 ; Vol. 121, No. 3. pp. 527-533.
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N2 - F-Actin treated with a half molar equivalent of the heterobifunctional cross-linker, 4-bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE), produced a population of actin oligomers containing 2-14 or more protomers and a significant amount of uncrosslinked actin protomers. The crosslinking reaction is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an adjacent protomer on the genetic helix of F-actin, in a similar; manner to N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers were found to form filaments that sediment after high-speed centrifugation, even in a buffer of low ionic strength [G-buffer: 2 mM tris-hydroxymethylaminomethane (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.3 mM NaN3, 5 mM 2-mercaptoethanol] where affinity between actin protomers usually becomes weak, leading to the depolymerization of native F-actin. By performing the crosslinking reaction in the presence of an environment-sensitive fluorescent label, 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which competes with BNBA-SE for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were obtained which were also stable in G-buffer. Fluorometric analysis of actin labeled with acrylodan (prodan-actin) in these depolymerization-resistant filaments suggested that the molecular environment around Cys 374 of actin protomers is similar to that of actin protomers in native F-actin in F-buffer (G-buffer plus 0.1 M KCl and 1 mM MgCl2). When G-actin labeled with acrylodan was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled actin was incorporated into filaments that were resistant to depolymerization in G-buffer. The emission spectrum of the prodan-actin in these filaments measured in G-buffer was almost identical to that of prodan-actin in native F-actin in F-buffer. Our interpretation of this result is that actin protomers are locked into an F-actin-like conformation in the depolymerization-resistant filament by the subunit interactions they make with crosslinked oligomers. We also present evidence which suggests that the depolymerization rate in G-buffer of filaments made with crosslinked oligomers is much slower than that of native actin because the ends of the depolymerization-resistant filaments are capped with crosslinked oligomers.

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