[8] Photoaffinity guanosine 5′-triphosphate analogs as a tool for the study of GTP-binding proteins

This chapter discusses photoaffinity guanosine 5'-triphosphate (GTP) analogs as a tool for studying GTP-binding proteins. Guanine nucleotide-binding proteins (G proteins) mediate myriad cellular functions from cell growth and division to protein synthesis and secretion. Heterotrimeric G proteins couple extracellular receptors to a variety of intracellular events, including the generation of second messengers and the transport of molecules across the cell membrane. The binding of GTP creates an activated G protein, and, once activated, the heterotrimeric G protein transmits and amplifies the message of a hormone or neurotransmitter through the invocation of a variety of intracellular processes. The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems, where purified components appear to reconstruct certain aspects of the receptor-G-protein coupling process. To study the relationship among G proteins, receptors, and effector molecules in complex systems, such as membranes or permeable cells, it is necessary to design probes which will allow, selectively, the examination of G-protein activation. One such probe is the hydrolysis-resistant, photoaffinity GTP analog P3-(4-azidoanilido)-P1-guanosine 5'-triphosphate. The chapter describes usefulness of this compound for both the examination of G-protein action in complex system and for the purification of G-protein.