Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Protein Sci. 2009 Nov;18(11):2326-35. doi: 10.1002/pro.243.
Crystal structures of Galpha(i) (and closely related family member Galpha(t)) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Galpha(t) exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Galpha(i), which exhibits higher basal exchange and a disordered Switch II region in Galpha(i)GDP structures. Using purified Galpha(i) and Galpha(t), we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Galpha proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GalphaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-pi interaction between tryptophan and arginine residues in the Switch II of Galpha(i) family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Galpha(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 Galpha proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
G 蛋白α亚基（包括与其密切相关的家族成员 Gαt）的晶体结构揭示了我们目前对 G 蛋白结构的了解，包括在开关区域发生的变化。与 Gαi 不同，Gαt 表现出低基础（非催化）核苷酸交换率和 GDP 结合状态下有序的开关 II 区，而 Gαi 则表现出更高的基础交换率和 GαiGDP 结构中无序的开关 II 区。使用纯化的 Gαi 和 Gαt，我们检测了这些蛋白的固有色氨酸荧光，这反映了与 G 蛋白激活和失活相关的构象变化。除了预期的色氨酸荧光强度增强外，GαGDP 蛋白的激活还伴随着色氨酸发射最大值的适度但显著的红移。我们确定了 G 蛋白α亚基家族蛋白开关 II 中的色氨酸和精氨酸残基之间的阳离子-π相互作用，该相互作用介导了发射最大值的观察到的红移。此外，Gαi 的氨基端豆蔻酰化导致 GTPase 结构域中色氨酸残基的极性降低，这与豆蔻酰化的氨基末端与 G 蛋白的 GTPase 结构域之间的相互作用一致。这些结果揭示了在溶液中 G 蛋白激活和失活时发生的构象变化的独特见解。
