Divalent cation binding to the high- and low-affinity sites on G-actin

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Dynamics

Background:

• Skeletal muscle G-actin is crucial for muscle contraction.
• Understanding metal ion interactions with G-actin is key to elucidating its functional mechanisms.
• Previous models proposed specific mechanisms for G-actin isomerization upon divalent cation binding.

Purpose of the Study:

• To characterize metal ion binding sites on skeletal muscle G-actin.
• To investigate the mechanism of G-actin isomerization induced by divalent cations.
• To explore the role of different divalent cations (Ca2+, Mg2+, Cd2+, Mn2+) in actin conformation.

Main Methods:

• Equilibrium dialysis using 45Ca2+ to quantify calcium binding.
• Kinetic measurements of fluorescence changes in N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin.
• Analysis of reaction progress curves using nonlinear regression fitting of kinetic simulations.

Main Results:

• Identified two classes of Ca2+ binding sites on G-actin: one high-affinity (Kd < 1 microM) and three moderate-affinity (Kd = 18 microM).
• Observed Mg2+-induced fluorescence enhancement supporting a mechanism where Ca2+ is replaced by Mg2+ at moderate sites, followed by slow actin isomerization.
• Demonstrated that actin isomerization occurs independently of Ca2+ release from the high-affinity site.
• Showed complex fluorescence changes with metal chelators and observed similar fluorescence changes with Cd2+ or Mn2+ as with Mg2+.

Conclusions:

• G-actin possesses distinct high- and moderate-affinity cation binding sites.
• Actin isomerization is triggered by Mg2+ binding at moderate sites and is independent of high-affinity Ca2+ release.
• Divalent cations induce subtle, cation-specific conformational changes in actin.
• A robust method for analyzing kinetic reaction progress curves was developed.