Crystal structures of Ga i (and closely related family member Ga t ) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Ga t exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Ga i , which exhibits higher basal exchange and a disordered Switch II region in Ga i GDP structures. Using purified Ga i and Ga t , we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Ga proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-p interaction between tryptophan and arginine residues in the Switch II of Ga i family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Ga i resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Ga proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
GPCRs catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to participate in anchoring G i proteins to the membrane, the role of myristoylation in regards to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Gα proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A.M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Gα i1 amino terminus during receptor-mediated G i activation has not been reported. Using site-specific fluorescent labeling of individual residues along a stretch of the Gα il amino terminus, we found specific changes in the environment of these residues upon interaction with activated receptor and following GTPγS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gβγ binding interface. The receptor-dependent fluorescent changes are consistent with a myristoylated amino terminus in close proximity to the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPγS binding). This work demonstrates that the myristoylated amino terminus of Gα il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.GPCRs act as GEFs for heterotrimeric G proteins, catalyzing nucleotide exchange on Gα subunits, which is the rate-limiting step in G protein activation. Agonist-mediated activation of GPCRs is coupled to GDP release by a mechanism which can be probed using biophysical techniques. While crystal structures for a number of G proteins and their respective subunits (1-7), as well as those of the prototypical GPCR, rhodopsin (8,9), and more recently the β2-adrenergic receptor (10), reveal much of what we know about the structure and function of these proteins, there is currently no high-resolution structure of an activated receptor in complex with its cognate G protein. Thus, biophysical and biochemical studies provide valuable insights regarding the structural determinants and dynamics of G protein activation. A functional heterotrimer is known to be essential for receptor-mediated G protein activation; the amino-terminal region of Gα directly participates in heterotrimer formation, facilitating high affinity Gα-βγ binding (11,12). † This work supported by NIH grant EY06062.* To whom correspondence should be addressed. Heidi E. Hamm, Department of Pharmacology, Vanderbilt University Medical School, 23rd Ave. South at Pierce, Nashville, Tennessee, 37232, Phone:(615) Fax: (615) The amino terminus of Gα (as well as the C terminus (13,14) and α4-β6 loop (15)) have been implicated...
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