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Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1
N Bettache1, R Bertrand, R Kassab
1Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U249, Université de Montpellier I, France.
Abstract:
We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Maleimidobenzoyl-G-actin (MBS-actin), a modified G-actin, retains native actin properties and polymerizes under specific conditions. MBS-actin also activates Mg2(+)-ATPase and inhibits DNase I, similar to native actin.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- G-actin is the globular monomeric form of actin, essential for muscle contraction and cell motility.
- Actin polymerization into F-actin is a dynamic process regulated by various factors, including ions and binding proteins.
- Myosin subfragment 1 (S-1) is a key motor protein that interacts with actin filaments.
Purpose of the Study:
- To investigate the structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a novel cross-linked G-actin derivative.
- To determine if MBS-actin polymerization is affected by salt and myosin subfragment 1 (S-1).
- To assess the functional similarities between MBS-actin and native G-actin in terms of polymerization, ATPase activation, and DNase I inhibition.
Main Methods:
- Circular dichroism (CD) spectroscopy to analyze alpha-helix content.
- 45Ca2+ binding assays to measure calcium content.
- Intrinsic fluorescence spectroscopy to monitor protein conformational changes.
- Polymerization assays under specific salt conditions (KCl, MgCl2).
- Enzyme activity assays for Mg2(+)-ATPase and DNase I inhibition.
Main Results:
- MBS-actin exhibited identical far-ultraviolet CD spectra and alpha-helix content compared to native G-actin.
- 45Ca2+ measurements and EDTA treatment revealed similar tightly bound Ca2+ content and fluorescence properties as native G-actin.
- MBS-actin polymerized at a critical monomer concentration of 1.6 mg/mL under specific salt conditions (100 mM KCl + 5 mM MgCl2).
- The resulting MBS-F-actin activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin.
- MBS-G-actin displayed DNase I inhibitor activity comparable to native G-actin and was unaffected by covalent conjugation to S-1.
Conclusions:
- MBS-actin is a structurally and functionally comparable derivative of native G-actin.
- MBS-actin polymerization can be induced under specific ionic conditions, similar to native G-actin.
- The modified actin derivative retains key functional interactions with myosin subfragment 1 and DNase I.