Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.
Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.
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DOI：
10.1021/bi201411c
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发表时间：
2011-12-13
期刊：
影响因子：
2.9
通讯作者：
Green, David F.
中科院分区：
文献类型：
--
作者：
Patsalo, Vadim;Raleigh, Daniel P.;Green, David F.
蓝病毒蛋白-N（CVN）是一种11 kDa的假对称蓝细菌凝集素，已被证明通过与病毒包膜糖蛋白gp 120表面上的高甘露糖寡糖结合来抑制人类免疫缺陷病毒（HIV）的感染。在这项工作中，我们描述了合理设计的CVN变体，稳定蛋白质折叠，同时保持高亲和力和选择性的聚糖目标。使用Poisson-Boltzmann计算和蛋白质重新包装算法来选择蛋白质核心中的稳定突变。通过用脂肪族基团取代Ser 11、Ser 20和Thr 61的埋置极性侧链，相对于先前表征的稳定化突变体，我们将CVN稳定化了近12 °C以对抗热变性，并且稳定化了1 μ m的GuaHCl以对抗化学变性。聚糖微阵列结合实验证实，碳水化合物结合的特异性谱不受突变的干扰，并且对于所有变体都是相同的。特别是，变体选择性结合含有Manα（1→2）Man键的聚糖，这是CVN的已知最小结合单位。我们还报告了慢变性动力学的CVN，并表明，它们可以复杂的热力学分析，特别是，展开的CVN不能被描述为一个固定的两态过渡。需要准确的热力学参数来描述CVN复杂的自由能景观，我们提供了更新的CVN展开值。
Cyanovirin-N (CVN) is an 11-kDa pseudo-symmetric cyanobacterial lectin that has been shown to inhibit infection by the Human Immunodeficiency Virus (HIV) by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work we describe rationally-designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson–Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 m of GuaHCl against chemical denaturation, relative to a previously-characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations, and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.
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影响因子：
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