Molecular Determinants of the Reversible Membrane Anchorage of the G-Protein Transducin

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Abstract
Transducin is a heterotrimer formed by a fatty acylated α-subunit and a farnesylated βγ-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated α-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the α(GDP)-subunit, the βγ-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 ± 0.3) × 106 M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 ± 0.5) × 106 M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 ± 0.3) × 106 M-1 (reflecting a major contribution of the α(GDP)-subunit with only a minor contribution from the βγ-complex). The apparent binding constant of the α(GTP)-subunit is distinctly smaller than that of the α(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2−3 cationic residues for the βγ-complex. The α-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.