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首页> 外文期刊>Biochemistry >Stabilization of an Intermediate Activation State for Transducin by a Fluorescent GTP Analogue
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Stabilization of an Intermediate Activation State for Transducin by a Fluorescent GTP Analogue

机译：通过荧光GTP类似物稳定转导蛋白的中间激活状态

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The GTP-binding protein (G protein),transducin,serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin.To better understand the structure-function characteristics of transducin activation,we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector,the cyclic GMP phosphodiesterase (PDE).In this study,we describe the characterization of a fluorescently labeled GTP analogue,BODIPY-FL GTPgammaS (BOD-GTPgammaS),that binds to the alpha subunit of transducin (alpha_T) in a rhodopsin- and Gbetagamma-dependent manner,similar to the binding of GTP or GTPgammaS,with an apparent dissociation constant of 100 nM.The rhodopsin-dependent binding of BOD-GTPgammaS to alpha_T is slow,relative to the rate of binding of GTPgammaS,particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha_T subunits.This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha_T-BOD-GTPgammaS complexes,relative to their rates of dissociation from alpha_T-GTPgammaS.The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha_T,indicating that only a subtle movement of the Switch 2 domain on alpha_T accompanies the binding of this GTPgammaS analogue.Nevertheless,the BOD-GTPgammaS-bound alpha_T subunit is able to bind with high affinity to the recombinant,purified gamma subunit of PDE (gammaPDE) labeled with 5-((((2-iodoacetyl)-amino)ethyl)amino)naphthalene-l-sulfonic acid (IAEDANS (K_d approx=13 nM)),as well as bind to and stimulate the activity of PDE,albeit less efficiently compared to alpha_T-GTPgammaS.Taken together,these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha_T,and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha_T in this conformational state with its biological target/effector.
机译：GTP结合蛋白（G蛋白）转导蛋白通过视紫质视紫红质对GTP结合（激活）/ GTP水解（失活）周期的严格调节，成为脊椎动物视觉中的关键分子开关。为了更好地了解结构转导蛋白激活的功能特征，我们已着手鉴定与该G蛋白的鸟嘌呤核苷酸结合位点结合并保持其与其特异性细胞靶标/效应子，环GMP磷酸二酯酶（PDE）相互作用的能力的光谱探针。在这项研究中，我们描述了荧光标记的GTP类似物BODIPY-FL GTPgammaS（BOD-GTPgammaS）的表征，该蛋白以视紫红质和Gbetagamma依赖性的方式与转导蛋白（alpha_T）的α亚基结合，类似于结合GTP或GTPgammaS的结合速率，表观解离常数为100 nM.BOD-GTPgammaS与α_T视紫红质的结合较慢，相对于GTPgammaS的结合速率，特别是在视紫红质必须起催化作用以刺激BOD-GTPgammaS与GDP在多个alpha_T亚基上交换的情况，这反映了视紫红质和Gbetagamma从alpha_T-BOD-GTPgammaS复合物中解离的速度较慢BOD-GTPgammaS的结合不会改变alpha_T的固有色氨酸荧光，这表明只有Switch 2域在alpha_T上的微妙移动才伴随着该GTPgammaS类似物的结合。尽管如此，BOD-GTPgammaS结合的alpha_T亚基。能够以高亲和力与标记有5-（（（（2-碘乙酰基）-氨基）乙基）氨基）萘-1-磺酸（IAEDANS（K_drox = 13 nM）），以及与PDE的结合和刺激其活性，尽管与alpha_T-GTPgammaS相比效率较低。综上所述，这些发现表明BOD-GTPgammaS与转导素的结合导致其采用PDE。独特的构象，似乎位于alpha_T的无活性和完全活性状态之间，该荧光核苷酸类似物可用作报告基团，以表征此构象状态下的alpha_T与生物学靶标/效应子的相互作用。

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