Xu Guozhong, Chance Mark R
Department of Physiology & Biophysics, Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461-1602, USA.
Anal Chem. 2005 Jul 15;77(14):4549-55. doi: 10.1021/ac050299+.
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 detectable 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 detectable. 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 approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.
羟基自由基介导的蛋白质足迹法是一种便捷且灵敏的技术，可用于绘制蛋白质的溶剂可及表面，并研究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了所有20种（常见）氨基酸侧链以及胱氨酸形成易于检测产物的反应活性和倾向。尽管我们之前已报道过其中许多残基的氧化情况，但本研究对反应活性较低的残基进行了详细考察，并更好地界定了它们在羟基自由基介导的足迹实验中的用途。对所有20种氨基酸酰胺以及胱氨酸和几种三肽进行γ射线辐照，通过电喷雾质谱分析产物，并测量修饰的速率常数。基于归一化至同时进行放射性标记作为内标的苯丙氨酸或脯氨酸的损失速率的质谱信号损失，比较氨基酸侧链的反应活性。通过这种方式，可以确保对相对速率进行准确的定量。获得了氨基酸侧链的反应活性顺序为：半胱氨酸＞甲硫氨酸＞色氨酸＞酪氨酸＞苯丙氨酸＞胱氨酸＞组氨酸＞亮氨酸、异亮氨酸＞精氨酸、赖氨酸、缬氨酸＞丝氨酸、苏氨酸、脯氨酸＞谷氨酰胺、谷氨酸＞天冬氨酸、天冬酰胺＞丙氨酸＞甘氨酸。丙氨酸和甘氨酸在典型实验中的反应活性极低，无法用作有用的探针，天冬氨酸和天冬酰胺似乎也不太可能有用。尽管丝氨酸和苏氨酸比已知可用作有用探针的脯氨酸更具反应活性，但其氧化产物不易检测。因此，在典型实验中，20种侧链（加上胱氨酸）中的14种最有可能是有用的。由于这些残基约占典型蛋白质序列的65%，因此足迹法能够很好地覆盖蛋白质侧链的反应活性。
