| G protein cycling requires dissociation and re-association of Gα and Gβγ subunits, therefore the interaction between these subunits is a critical one. In the inactive heterotrimer, the amino terminus is seen as an ordered α-helix in contact with Gβγ. This amino terminal interaction with Gβγ buries about a third of the total surface area involved in binding of Gα to Gβγ. In the absence of Gβγ, little is known about the structure of the amino terminus of Gα. In addition, myristoylation of Gα subunits adds another level of complexity to G protein signaling.; We have devised methods to examine the environment and mobility of specifically labeled Gαi residues using fluorescence and electron paramagnetic spectroscopy upon cycles of activation and deactivation. Studies were performed on nine different sites on the amino terminus of myristoylated and non-myristoylated Gαi proteins, in both the inactive (GDP bound and Gβγ bound) and activated states. These studies revealed the amino terminus of the non-myristoylated Gαi proteins have very little structure in the absence of Gβγ. This is in contrast to the myristoylated proteins, which adopt a highly ordered and immobile structure, even in the absence of Gβγ. Fluorescence results revealed dramatic changes in the polarity of the environment detected by fluorescent probes attached to amino terminal residues upon activation, indicative of a GTP dependent myristoyl switch for the amino terminus of Gα proteins. The high degree of immobility seen in fluorescence anisotropy and EPR spectra, combined with increase in hydrophobic environment seen in steady state fluorescence studies altogether suggest the possibility of an intramolecular binding site on the surface of the Gα molecule for the myristoylated amino terminus. Together, these mechanisms may regulate movements of Gα proteins on and off membranes and may provide a mechanism for the spatial regulation of G protein signaling. |
