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The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system.

Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature of our software and ready availability of freeware tools used for its development will facilitate its adaptation and further development. The software is distributed under GPLv3 licence and is available at http://www.psi.ch/lbr/aascan.