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首页> 外文期刊>Biochemistry >INVESTIGATION OF THE GTP-BINDING GTPASE CYCLE OF CDC42HS USING EXTRINSIC REPORTER GROUP FLUORESCENCE
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INVESTIGATION OF THE GTP-BINDING GTPASE CYCLE OF CDC42HS USING EXTRINSIC REPORTER GROUP FLUORESCENCE

机译：利用外源报告基团荧光研究CDC42HS的GTP结合GTP酶循环

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The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)am (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of similar to 32 Angstrom for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.
机译：这些研究的总体目标是检查外部报道基因组荧光在监测Ras样GTP结合蛋白的GTP结合/ GTPase循环中的适用性。为此，我们在一个反应​​性赖氨酸上用对环境敏感的荧光团琥珀酰亚胺6-（[（7-硝基苯-2-氧杂-1,3-二唑-4-基）am]（sNBD）标记了GTP结合蛋白Cdc42Hs。残留物。我们发现sNBD标记的Cdc42Hs在545 nm处发生荧光增强，而Cdc42Hs将结合的GDP交换为GTP。然后，在GTP水解后，这种增强作用会完全逆转。 Cdc42Hs的特异性GTPase激活蛋白Cdc42Hs-GAP强烈刺激了545 nm处荧光增强的逆转速率，与其完全催化Cdc42Hs的GTPase反应的能力一致。相反，特定鸟嘌呤核苷酸交换因子（GEF）Cdc24强烈刺激了GTP结合所伴随的荧光增强，这与其刺激Cdc42Hs的GDP-GTP交换反应的能力一致。共振能量转移测量得出，sNBD部分和鸟嘌呤核苷酸结合位点被N-甲基蒽基-（Mant）dGDP或MantdGTP占据的距离接近32埃。综上所述，这些结果在Cdc42Hs分子上确定了一个构象敏感的报告位点，该位点与鸟嘌呤核苷酸结合位点相距一定距离，但仍为GTP结合，GTPase活性以及关键调节蛋白的相互作用提供了高度敏感的监测器。

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