Purchase this article with an account.
M. Downs, O. Kisselev; Membrane–Binding Properties of Transducin Beta–Gamma Dimers . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1696.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Interactions between light–activated rhodopsin and the G–protein transducin is accompanied by the conformational regulatory switch in the C–terminal region of Gt–gamma subunit. This domain Gt–gamma(60–71) is in the extended conformation in the heterotrimer, but it adopts an amphipathic helical conformation in the R*–bound form. (Kisselev, O.G. and Downs, M.A. (2003) Structure, Cell Press 11, 367–373). The Gt–gamma(60–71) helix appears to run in parallel to the plane of the membrane and it may contain membrane binding elements in addition to the amino acid side chains involved in R*–binding. Purpose: To investigate contribution of individual side chains into membrane binding we have used Alanine and other substitutions in the Gt–gamma(60–71) region and examined the effects of the replacements on the ability of the mutant proteins to interact with lipid membranes. Methods: Multiple mutations have also been introduced. The mutant Gt–gamma proteins have been co–expressed with histidine labeled Gt–beta subunits in SF9 insect cells to form functionally active dimers, as determined by their ability to interact with R* after reconstitution with purified Gt–alpha. Results: The extent of membrane binding for each mutant dimer was measured by quantitative immunoblotting and compared to the wild–type protein. Conclusions: The results shed light on the importance of charged and hydrophobic amino–acid side chains in the C–terminal region of Gt–gamma and help to model the rhodopsin–transducin interface. This work is supported in part by NIH and Foundation for Research to Prevent Blindness.
This PDF is available to Subscribers Only