Institute of Medical Virology, University of Tübingen, Elfriede-Aulhorn-Str. 06, D-72076 Tuebingen, Germany.
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 03, D-06120, Halle/Saale, Germany.
Biol Chem. 2019 Mar 26;400(4):513-522. doi: 10.1515/hsz-2018-0311.
The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.
乳头瘤病毒的次要衣壳蛋白 L2 在病毒进入过程中表现出多种功能，包括与膜相互作用。由于其高度聚集的倾向，关于该蛋白的信息很少。我们确定了合适的条件来生产功能性人乳头瘤病毒（HPV）16 L2 蛋白，从而为 L2 蛋白的结构和生化信息以及病毒进入的机制细节提供了广泛的体外分析机会。我们在大肠杆菌中作为包涵体生产了高危 HPV 16 的 L2 蛋白，并在变性条件下对其进行了纯化。连续的缓冲液筛选为 16L2 的生物物理特性分析提供了合适的条件。重折叠蛋白的分析超速离心显示出均一的单体物种。此外，重折叠的 16L2 显示出二级结构元件。包括推测的跨膜区域的 16L2 的 N 端区域具有α-螺旋特征。然而，总的来说 16L2 似乎很大程度上没有结构。重折叠的 16L2 能够结合 DNA，表明在重折叠的 16L2 中，假定的 DNA 结合区域是可及的。此外，重折叠的蛋白在中性 pH 下与脂质体膜相互作用，推测是通过跨膜区域相互作用，而没有结构变化。这表明 16L2 可以通过预先存在的结构特征最初与膜相互作用。
