首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Segmental dynamics of the cytoplasmic domain oferythrocyte band 3 determined by time-resolved fluorescence anisotropy:sensitivity to pH and ligand binding.
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Segmental dynamics of the cytoplasmic domain oferythrocyte band 3 determined by time-resolved fluorescence anisotropy:sensitivity to pH and ligand binding.

机译:胞质结构域的节段动力学通过时间分辨荧光各向异性确定的红细胞带3:对pH和配体结合的敏感性。

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摘要

Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of approximately 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (tau 1c and tau 2c) and two limiting anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity and r2infinity. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence.Significant changes in cdb3 segmental motion occurred upon interactions with thesmall ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymesknown to bind to the N terminus of band 3. These time-resolved fluorescencemeasurements of the nanosecond segmental dynamics of a labeled membrane proteinprovide evidence for the sensitivity of cdb3 conformation to ligand binding andsuggest long-range structural communication through cdb3.
机译:红细胞膜与其骨架之间的相互作用主要通过细胞骨架成分与构象敏感结构(带3的胞质结构域(cdb3))的结合来介导。为了检查cdb3的纳秒分段运动,通过荧光素马来酰亚胺在cdb3中拟议的铰链附近的Cys-201选择性标记了条带3,围绕该铰链发生了pH依赖的构象变化。通过平行采集频域微荧光法测量分离的形式和剥离的红细胞膜中标记的cdb3的时间分辨各向异性。样品的单组分荧光素寿命约为4 ns。多频相位和调制数据(5-20​​0 MHz)非常适合包含两个相关时间(τ1c和tau 2c)和两个极限各向异性(r1无穷大和r2无穷大)的分段运动模型。蛋白酶切割和变性样品的测量结果表明,tau 1c(100-150 ps)对应于结合的荧光素的快速旋转,tau 2c(2-5 ns)对应于cdb3的分段运动。如非零r1infinity和r2infinity所量化,这两个运动均受到阻碍。分段运动的强pH依赖性与通过固有色氨酸荧光测量的cdb3构象的pH依赖性相关。cdb3节段运动的显着变化发生在与2,3-双磷酸甘油酸和钙的小配体和几种糖酵解酶已知与3带的N末端结合。这些时间分辨的荧光标记的膜蛋白的纳秒节段动力学测量为cdb3构象对配体结合的敏感性提供证据,并建议通过cdb3进行远程结构通信。

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