Hirudin is a bioactive small protein that binds thrombin to interrupt the blood clotting cascade. It contains an ordered and a disordered (IDR) region. Conjugating with polyethylene glycol (PEGylation) is an important modification of biopharmaceuticals to improve their lifetime and retention. Here, we studied by molecular dynamics (MD) simulation how hirudin P18 and its PEGylated variant differ in their structural flexibility depending on binding to thrombin and charge screening by NaCl. We also compare with glycated hirP18 and the hirV1 variant to assess effects of different polar attachments and sequence variability. First, we synthesized unlabeled and PEG-labeled hirP18 followed by an activity assay to ascertain that the peptide-PEG conjugate retains anticoagulant activity. Next, we carried 16 different microsecond MD simulations of the different proteins, bound and unbound, for 2 sequences and different salt conditions. Simulations were analyzed in terms of scaling exponents to study the effect of ionic strength on hirudin size and solvent-exposed surface area. We conclude that charge patterning of the sequence and the presence of arginine are 2 important features for how PEG interacts with the protein folded and intrinsically disordered regions. Specifically, PEG can screen end-to-end electrostatic interactions by “hiding” a positively charged region of hirudin, whereas hirV1 is less sticky than hirP18 due to different PEG-hirudin hydrophobic interactions and the presence of an arginine in hirP18. Conjugation with either PEG or a glycan significantly reduces solvent-exposed area of hirudin, but PEG interacts more efficiently with surface residues than does glycan due to its narrower chain that can fit in surface grooves, and alternation of polar (oxygen) and nonpolar (CH2-CH2) groups that interact favorably with charged and hydrophobic surface patches.

中文翻译：

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聚乙二醇对水蛭素结构和动力学的环境依赖性影响


水蛭素是一种具有生物活性的小蛋白，可与凝血酶结合以中断血液凝固级联反应。它包含一个有序区域和一个无序 （IDR） 区域。与聚乙二醇偶联（聚乙二醇化）是生物制药的重要改性，可延长其使用寿命和保留率。在这里，我们通过分子动力学 （MD） 模拟研究了水蛭素 P18 及其聚乙二醇化变体的结构灵活性如何根据与凝血酶的结合和 NaCl 的电荷筛选而有所不同。我们还与糖化 hirP18 和 hirV1 变体进行比较，以评估不同极性连接和序列变异性的影响。首先，我们合成了未标记和 PEG 标记的 hirP18，然后进行活性测定以确定肽-PEG 偶联物保留了抗凝活性。接下来，我们针对 2 个序列和不同的盐条件，对不同蛋白质（结合和未结合）进行了 16 个不同的微秒 MD 模拟。根据缩放指数分析模拟，以研究离子强度对水蛭素大小和溶剂暴露表面积的影响。我们得出结论，序列的电荷模式和精氨酸的存在是 PEG 如何与蛋白质折叠和固有无序区域相互作用的两个重要特征。具体来说，PEG 可以通过“隐藏”水蛭素的带正电区域来筛选端到端静电相互作用，而 hirV1 的粘性低于 hirP18，因为 PEG-水蛭素疏水相互作用不同，并且 hirP18 中存在精氨酸。 与 PEG 或游离寡糖结合可显著减少水蛭素的溶剂暴露面积，但 PEG 与表面残基的相互作用比游离寡糖更有效，因为它的链较窄，可以进入表面凹槽，并且极性（氧）和非极性 （CH2-CH2） 基团交替，与带电和疏水的表面斑块有利地相互作用。