Molecular determinants of the reversible membrane anchorage of the G-protein transducin

被引:46
|
作者
Seitz, HR
Heck, M
Hofmann, KP
Alt, T
Pellaud, J
Seelig, A
机构
[1] Univ Basel, Bioctr, Dept Biophys Chem, CH-4056 Basel, Switzerland
[2] Humboldt Univ, Inst Med Phys & Biophys, Med Fak Charite, Berlin, Germany
关键词
D O I
10.1021/bi990298+
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated beta gamma-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 alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the beta gamma-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 K-app = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), K-app = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and K-app = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the beta gamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the beta gamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.
引用
收藏
页码:7950 / 7960
页数:11
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