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首页> 外文期刊>Biochemistry >Fluorophore-assisted light inactivation of calmodulin involves singlet-oxygen mediated cross-linking and methionine oxidation
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Fluorophore-assisted light inactivation of calmodulin involves singlet-oxygen mediated cross-linking and methionine oxidation

机译：钙调蛋白的荧光团辅助光灭活涉及单线态氧介导的交联和蛋氨酸氧化

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Fluorophore-assisted light inactivation (FALI) permits the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding target protein sensitivity. To permit the rational inactivation of targeted proteins, we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins, regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (RASH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between similar to 7 and 40 angstrom. In addition, we observed a loss of histidines, as well as zero-length cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces.
机译：荧光团辅助的光灭活（FALI）允许标签蛋白的靶向失活，并且当与细胞可渗透的多用途亲和探针（MAPs）一起使用时，在鉴定生理功能方面具有重要优势，因为可以通过空间和时间控制实现目标蛋白的失活。但是，FALI的可靠应用（也称为荧光素衍生物的生色团辅助光灭活（CALI））由于缺乏有关靶蛋白敏感性的机械信息而受到限制。为了使靶蛋白合理失活，我们已经确定了氧化性物质和特定氨基酸对钙调节蛋白钙调蛋白（CaM）修饰的敏感性，钙调蛋白钙调蛋白与许多必需蛋白一样，通过与大分子的可逆结合来调节信号转导。靶蛋白的数量。继共价和刚性连接的4'，5'-双（1,3,2-二硫代呋喃-2-基）荧光素（RASH）到螺旋A上，我们发现了内源性蛋氨酸对其相应蛋氨酸的光依赖性氧化修饰亚砜。甲硫氨酸氧化的初始速率与表面可及性相关，并且对结合的荧光团与各个甲硫氨酸之间的距离不敏感，后者之间的差异在7到40埃之间。另外，我们观察到组氨酸的损失，以及与对应伴侣的零长度交联，所述对应伴侣对应于平滑肌球蛋白轻链激酶和ryanodine受体的CaM结合位点。我们的结果为使用FALI抑制许多信号蛋白（如CaM）通常在结合界面上存在蛋氨酸的功能提供了蛋白质组学筛选的依据。

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