Receptor binding site-specific mutation

There are two basic considerations when attempting SDM. One is to determine the amino acids that should be mutated and the other is to decide what to replace them with. The first question is, of course, dependant upon information gathered from previous experimentation in order to target residues that are appropriate. Such information may be derived from biochemical techniques. For instance, in our binding site studies, we have specifically mutated amino acids that had previously shown to be covalently labeled by photoactive ligands. Additionally, we have used comparisons between the sequences of different receptor subunits that correlate with receptor function to identify domains of interest. Chimeragenesis, the technique described in the first half of this chapter, can provide important information in this regard. Obviously, those proteins for which a detailed structural model is available will lend themselves to more rational substitutions. 

Despite the extensive data describing the receptor-binding surface on G proteins, much less is known about the specific point-to-point interactions between these proteins. Generally, the membrane-proximal sections of IC 2, 3, and 4 form the primary interaction surface on the receptor. Additional contact sites in the transmembrane helices may also be formed as the Gy C-terminus enters deeply into the pocket formed in the cytoplasmic face of the receptor on activation (Janz and Farrens, 2004). Peptides corresponding to IC 2, 3, and 4 block the stabilization of Meta II by Gat (Konig et at., 1989 Marin et at., 2000). A reverse substitution approach, where each intracellular loop was replaced with polyalanine sequences and then individual alanine residues were mutated back to the native amino acid, demonstrated that residues adjacent to the transmembrane helices in these loops were the most important for G protein activation (Natochin et at., 2003). 

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