Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting
Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting
复制标题
DOI：
10.1021/ac050299
复制
发表时间：
2005-07-15
影响因子：
7.4
通讯作者：
Chance, MR
中科院分区：
文献类型：
--
作者：
Xu, GZ;Chance, MR
羟基自由基介导的蛋白质足迹是一种方便且灵敏的技术，用于绘制蛋白质的溶剂可及表面并检查生物组装体的结构和动力学。在本研究中，使用各种标准直接比较所有 20 种（常见）氨基酸侧链以及胱氨酸的反应性和形成易于检测的产物的趋势。尽管我们之前报道过许多这些残留物的氧化，但这项研究包括对反应性较低的残留物的详细检查，并更好地定义了它们在羟基自由基介导的足迹实验中的有用性。所有20种氨基酰胺以及胱氨酸和一些三肽均经过伽马射线照射，通过电喷雾质谱分析产物，并测量修饰的速率常数。氨基酸侧链的反应性根据其质谱信号损失进行比较，该损失归一化为同时放射分解以用作内标的 Phe 或 Pro 的损失率。通过这种方式，可以确保相对速率的准确定量。氨基酸侧链的反应性顺序为Cys>Met>Trp>Tyr>Phe>胱氨酸>His>Leu、Ile>Arg、Lys、Val>Ser、Thr、Pro>Gln、Glu>Asp、Asn>Ala>Gly。 Ala 和 Gly 的反应活性太低，无法在典型实验中用作有用的探针，而 Asp 和 Asn 也不太可能有用。虽然 Ser 和 Thr 比 Pro 更具反应性，而 Pro 是一种有用的探针，但它们的氧化产物不容易检测到。因此，20 条侧链中的 14 条（加上胱氨酸）似乎最有可能在典型实验中有用。由于这些残基与典型蛋白质的 65% 序列相似，因此足迹法可以很好地覆盖蛋白质的侧链反应性。
Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise similar to 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.