Engineering unnatural nucleotide specificity to probe G protein signaling.
G proteins comprise approximately 0.5% of proteins encoded by mammalian genomes. To date, there exists a lack of small-molecule modulators that could contribute to their functional study. In this report, we present the use of H-Ras to develop a system that answers this need. Small molecules that allow for the highly specific inhibition or activation of the engineered G protein were developed. The rational design preserved binding of the natural substrates to the G protein, and the mutations were functionally innocuous in a cellular context. This tool can be used for isolating specific G protein effectors, as we demonstrate with the identification of Nol1 as a putative effector of H-Ras. Finally, the generalization of this system was confirmed by applying it to Rap1B, suggesting that this method will be applicable to other G proteins.
Vincent
Fabien
F
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Cook
Silas P
SP
Johnson
Emmanuel O
EO
Emmert
Dana
D
Shah
Kavita
K
D000818:Animals; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes / Q000235:genetics* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D015398:Signal Transduction; D013379:Substrate Specificity
D015335:Molecular Probes; D009711:Nucleotides; D019204:GTP-Binding Proteins