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Domain structure in actin-binding proteins: expression and functional characterization of truncated severin
L Eichinger1, A A Noegel, M Schleicher
1Max-Planck-Institute for Biochemistry, Martinsried, Federal Republic of Germany.
The Journal of Cell Biology
|February 1, 1991
Summary
Severin, a calcium-activated actin-binding protein, has its severing and nucleating functions localized to two adjacent sites. A single N-terminal actin-binding site is sufficient for capping filaments.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Severin is a calcium-activated actin-binding protein from Dictyostelium discoideum.
- It exhibits actin filament severing, nucleation, and capping activities.
- Conserved domains suggest functional significance, similar to proteins like gelsolin.
Purpose of the Study:
- To map the functional domains responsible for severin's actin-binding activities.
- To determine the role of specific regions in calcium-dependent regulation.
Main Methods:
- Expression and purification of truncated severin polypeptides (DS362, DS277, DS177, DS151, DS117, DS111).
- Characterization of actin polymerization/depolymerization kinetics and G-actin binding.
- Investigation of calcium binding using 45Ca overlay and tryptophan fluorescence.
Main Results:
- Full-length severin (DS362) showed native calcium-dependent activities.
- Truncated forms revealed distinct domain functions: DS277 retained calcium-dependent severing/nucleation but had independent capping.
- N-terminal regions (DS111, DS117, DS151) were responsible for capping, with stabilization observed upon extension.
Conclusions:
- Severin possesses two adjacent actin-binding sites responsible for both severing and nucleation.
- A single N-terminal actin-binding site is sufficient for filament capping.
- Unlike gelsolin, severin's functional domains are primarily localized in close proximity.