Heat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance. Each of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1. The stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.

中文翻译：

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N端附近卷曲螺旋结构的稳定性影响黄单胞菌harpin蛋白的耐热性


耐热性是 harpins 的一个共同特征，harpins 是革兰氏阴性细菌中发现的一类蛋白质，这可能与卷曲螺旋 (CC) 结构的稳定性有关。 CC 结构是一种普遍存在的蛋白质折叠和组装基序，由相互缠绕形成超螺旋的 α 螺旋组成。具体而言，研究了来自黄单胞菌的四种选定的harpin蛋白（以下称为Hpa1）靠近N末端的CC结构的稳定性是否会影响它们的耐热特性。我们利用生物信息学方法预测Hpa1的结构，利用Hpa1的过敏反应（HR）诱导活性的表现和圆二色性（CD）光谱分析来检测Hpa1的CC结构的稳定性与耐热性之间的关系。四个选定的 Hpa1 中的每一个都有两个 α 螺旋区域，其中一个在其 N 端可以形成 CC 结构，另一个在其 C 端则不能形成 CC 结构。 CC 基序中涉及的重要氨基酸残基位于这些蛋白质表面的螺旋上，表明它们可能参与寡聚聚集体的形成，这可能是 harpins 引发 HR 及其高热稳定性的原因。增加或减少形成CC的可能性可以诱导更强的HR反应或消除高温处理后在烟草中诱导HR的能力。此外，虽然4个Hpa1突变体对Hpa1诱导HR影响不大，但其热稳定性显着下降。 α-螺旋含量随着温度的升高而增加，当温度达到200℃时，Hpa1的二级结构几乎完全变成α-螺旋。 此外，发现靠近N端的CC结构的稳定性与Hpa1的耐热性呈正相关。 CC结构的稳定性可能作为介导harpin蛋白耐热性的内在驱动力。我们的研究结果为解释harpin蛋白耐热性机制提供了新的见解，并为进一步harpin功能研究和结构修饰提供了理论基础。