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Membrane association and conformational change of palmitoylated G(o)alpha
1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China.
Abstract:
Bovine brain G(o)alpha was specifically palmitoylated in vitro. The apparent dissociation constant for depalmitoylated G(o)alpha (dG(o)alpha) was 0.273 microM, while that for palmitoylated G(o)alpha (pG(o)alpha) was 5.77 nM. The dissociation rate constant (K(21)) and dissociation half-life for dG(o)alpha were 8.4x10(-4) min and 825 min respectively, while no significant dissociation of pG(o)alpha was detected. The limiting membrane insertion pressures for pG(o)alpha and dG(o)alpha were 44.4 mN/m and 41.3 mN/m respectively. These data suggested that palmitoylation facilitated the membrane association of G(o)alpha. Conformational changes of dG(o)alpha and pG(o)alpha detected by monitoring fluorescence spectra and fluorescence quenching were significantly different after they were associated with the membrane. It was suggested that conformational changes of G(o)alpha upon membrane association might be related to regulation of G(o)alpha signaling by palmitoylation.
Insights
Palmitoylation, a lipid modification, enhances the membrane association of bovine G(o)alpha proteins. This modification alters protein conformation, potentially regulating G(o)alpha signaling pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- G(o)alpha is a heterotrimeric G protein alpha subunit found in bovine brain.
- Protein palmitoylation is a post-translational modification involving the addition of a palmitoyl group, often affecting protein localization and function.
- Understanding the role of palmitoylation in G(o)alpha function is crucial for elucidating cellular signaling mechanisms.
Purpose of the Study:
- To investigate the effect of palmitoylation on the membrane association of bovine G(o)alpha.
- To characterize the biophysical properties of palmitoylated and depalmitoylated G(o)alpha.
- To explore the conformational changes of G(o)alpha upon membrane association as influenced by palmitoylation.
Main Methods:
- In vitro palmitoylation of bovine G(o)alpha.
- Measurement of apparent dissociation constants (Kd) for palmitoylated (pG(o)alpha) and depalmitoylated (dG(o)alpha) forms.
- Determination of dissociation rate constants (K21) and half-lives.
- Measurement of limiting membrane insertion pressures.
- Analysis of conformational changes using fluorescence spectroscopy and quenching.
Main Results:
- Palmitoylation significantly increased the affinity of G(o)alpha for membranes, with a much lower dissociation constant for pG(o)alpha (5.77 nM) compared to dG(o)alpha (0.273 microM).
- pG(o)alpha showed minimal dissociation from membranes, unlike dG(o)alpha, which had a dissociation half-life of 825 minutes.
- Palmitoylation slightly increased the limiting membrane insertion pressure.
- Distinct conformational changes were observed for pG(o)alpha and dG(o)alpha after membrane association, detectable via fluorescence spectroscopy.
Conclusions:
- Palmitoylation is a key facilitator of G(o)alpha membrane association.
- The altered membrane interaction and conformational state of palmitoylated G(o)alpha suggest a regulatory role for this modification in G(o)alpha signaling.
- These findings provide insights into the molecular mechanisms by which palmitoylation modulates G protein function.