Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes
Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes
复制标题
荧光蛋白探针快速谷氨酸传感的动力学机制
DOI：
10.1016/j.bpj.2019.11.006
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发表时间：
2020
影响因子：
3.4
通讯作者：
Coates C
中科院分区：
文献类型：
--
作者：
Coates C
我们开发了基于细菌周质谷氨酸/天冬氨酸结合蛋白的探针，以内源荧光蛋白或合成荧光团作为谷氨酸结合的指示剂，用于研究谷氨酸结合的动力学机制。 iGluSnFR 变体称为 iGluh、iGlum 和 iGlul，涵盖广泛的 Kd-s（分别为 5.8μM、2.1 和 50 mM），而新型荧光标记指示剂 Fl-GluBP 的 Kd-s 为 9.7μM。所有探针的荧光响应动力学与涉及配体结合和apo或配体结合结合蛋白异构化的两步机制一致。尽管先前表征的超快指示剂 iGluu 和 iGluf 在限速异构化步骤中发生单相荧光增强，但此处描述的传感器都具有双相结合动力学，在谷氨酸结合和异构化步骤中均发生荧光增强。对于 iGlum 和 iGlul，数据表明结合前构象变化，随后是配体结合。相反，对于 iGluhand Fl-GluBP，谷氨酸结合之后是异构化。因此，揭示了结合位点周围单个氨基酸变化引入的结构异质性对与谷氨酸相互作用的动力学路径的影响。值得注意的是，首次检测到谷氨酸以扩散限制速率常数与 iGluhand Fl-GluBP 结合，暗示了通过谷氨酸结合极快速激活高度同源的 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的潜在机制。
We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGluh, iGlum, and iGlulcover a broad range ofKd-s (5.8μM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has aKdof 9.7μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGluuand iGlufhad monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlumand iGlul, the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGluhand Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion-limited rate constant to iGluhand Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologousα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding.