Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor
Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor
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DOI：
10.1074/jbc.m201164200
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发表时间：
2002-05-24
影响因子：
4.8
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Harikumar, KG;Pinon, DL;Miller, LJ
荧光是分析蛋白质结构和动力学的一种强大的生物物理工具。在这里，我们开发了两个新的CCK受体荧光探针，代表结构相关的多肽激动剂和拮抗剂。每个配体都有三个不同的荧光团之一(Alexa(488)，硝基苯并恶二唑基，或acrylowan)，在激素药效团外的氨基末端有类似的位置。所有的探针都与CCK受体特异性结合，亲和力很高，细胞内钙信号研究表明，经过化学修饰的多肽具有完全的生物活性。通过碘化物猝灭、荧光光谱、各向异性和寿命的测量来表征多肽-受体复合体中探针对激动剂和拮抗剂的荧光响应。这三种荧光指示剂对同一指示剂在与CCK受体结合的激动剂和拮抗剂多肽的类似位置上的环境差异提供了相同的见解。与类似的拮抗剂相比，每种激动剂的荧光更容易熄灭，表现出更低的各向异性(更高的探针迁移率)和更短的寿命。用非水解性GTP类似物处理激动剂占据的受体，使受体进入不活跃的低亲和力状态，并增加了探针荧光寿命，接近拮抗剂探针的观察值。这些数据与与受体激活相关的分子构象变化一致，该变化导致配体的氨基末端(位于跨膜片段6上方)从其某种程度上受保护的环境向水环境移动。
Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa(488), nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.