Designing point mutants to detect structural coupling in a heterotrimeric G protein alpha-subunit by NMR spectroscopy.
Designing point mutants to detect structural coupling in a heterotrimeric G protein alpha-subunit by NMR spectroscopy.
复制标题
设计点突变体以通过核磁共振波谱检测异源三聚体 G 蛋白 α 亚基中的结构耦合。
DOI：
10.1111/j.1751-1097.2008.00522.x
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发表时间：
2009
影响因子：
3.3
通讯作者：
Ridge,KevinD
中科院分区：
文献类型：
--
作者：
Abdulaev,NajmoutinG;Mao,Xiang;Ramon,Eva;Ngo,Tony;Mysliwy,Justyna;Marino,JohnP;Ridge,KevinD
为了更好地了解激活信号从G蛋白α亚单位(Gα)上的受体相互作用区传递到鸟核苷酸结合口袋的机制，我们产生并鉴定了Gα的突变形式，其开关II(Trp-207→Phe)和羧基末端(Phe-350→Ala)发生了变化。以前报道的用于高水平生产统一同位素标记的GTα/Gi1α嵌合体CHIT的细菌表达方法被成功地用于分离毫克量的15N标记突变蛋白。核磁共振分析表明，虽然两个突变体的Gdp/Mg~(2+)结合态与Chit的Gdp/Mg~(2+)结合态的总体构象相似，但在氟化铝存在下形成的“过渡/激活”态显示了野生型和突变型G-−亚基之间的明显差异，特别是在Trp-207→Phe突变体中Trp-254吲哚的1HN，15N交叉峰和在Phe-350→Ala突变体中Ala-350的1HN，15N交叉峰。与核磁共振数据一致的是，F350→Ala突变体的本征荧光增强，与GTα和Chit相似，在AlF4−存在下形成“跃迁/激活”状态，而Trp-207→Phe突变体的本征荧光减弱。这些结果表明，G-α中关键氨基酸位置的替换可以影响结构变化，这些变化可能会影响受体相互作用和Gdp/GTP交换。
To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (Gα) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of Gαwith alterations in switch II (Trp‐207→Phe) and the carboxyl‐terminus (Phe‐350→Ala). Previously reported bacterial expression methods for the high‐level production of a uniformly isotope‐labeled Gtα/Gi1αchimera, ChiT, were successfully used to isolate milligram quantities of15N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg2+‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg2+‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF4−) revealed distinct differences between the wild‐type and mutant Gαsubunits, particularly in the response of the1HN,15N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the1HN,15N cross‐peak for Ala‐350 in the Phe‐350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to Gtαand ChiT upon formation of the “transition/activated” state in the presence of AlF4−, whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in Gαcan effect structural changes that may compromise receptor interactions and GDP/GTP exchange.