Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Gelsolin is a key actin-binding protein involved in regulating actin cytoskeleton dynamics.
• Calcium (Ca2+) ions are known to modulate gelsolin's activity, but the precise structural transitions are not fully understood.

Purpose of the Study:

• To elucidate the detailed structural changes in gelsolin upon Ca2+-dependent activation.
• To identify the specific regions and residues involved in these transitions and their relation to gelsolin's functions.

Main Methods:

• Radiolytic protein footprinting using synchrotron radiation to assess solvent accessibility of peptide segments.
• Mass spectrometry and tandem mass spectrometry to identify oxidized residues and map structural changes.
• Ca2+ titration experiments to monitor changes in solvent accessibility as a function of Ca2+ concentration.

Main Results:

• Identified over 80 peptide segments, covering 95% of gelsolin, with varying solvent accessibility.
• Revealed a three-state Ca2+ activation process occurring at distinct Ca2+ concentrations (1-5 microM and 60-100 microM).
• Showed that Ca2+ binding unmasks F-actin-binding sites in subdomains S2 and S6 at low concentrations, and further exposes S4 residues at higher concentrations.

Conclusions:

• Gelsolin activation involves at least two major structural transitions dependent on Ca2+ concentration.
• The initial Ca2+ transition exposes F-actin binding sites, enabling capping and severing.
• The subsequent transition likely leads to a more extended conformation, facilitating additional functions of the fully activated protein.