Bourrelle-Langlois Maxime, Morrow Geneviève, Finet Stéphanie, Tanguay Robert M
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Intégrative et des Systémes (IBIS) and PROTEO, Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de Médecine, Québec, Canada.
IMPMC UMR7590, CNRS/Sorbonne-Universités, UPMC/IRD/MNHN Paris 6, Paris, France.
PLoS One. 2016 Sep 19;11(9):e0162233. doi: 10.1371/journal.pone.0162233. eCollection 2016.
We previously reported the in silico characterization of Synechococcus sp. phage 18 kDa small heat shock protein (HspSP-ShM2). This small heat shock protein (sHSP) contains a highly conserved core alpha crystalline domain of 92 amino acids and relatively short N- and C-terminal arms, the later containing the classical C-terminal anchoring module motif (L-X-I/L/V). Here we establish the oligomeric profile of HspSP-ShM2 and its structural dynamics under in vitro experimental conditions using size exclusion chromatography (SEC/FPLC), gradient native gels electrophoresis and dynamic light scattering (DLS). Under native conditions, HspSP-ShM2 displays the ability to form large oligomers and shows a polydisperse profile. At higher temperatures, it shows extensive structural dynamics and undergoes conformational changes through an increased of subunit rearrangement and formation of sub-oligomeric species. We also demonstrate its capacity to prevent the aggregation of citrate synthase, malate dehydrogenase and luciferase under heat shock conditions through the formation of stable and soluble hetero-oligomeric complexes (sHSP:substrate). In contrast, the host cyanobacteria Synechococcus sp. WH7803 15 kDa sHSP (HspS-WH7803) aggregates when in the same conditions as HspSP-ShM2. However, its solubility can be maintained in the presence of non-ionic detergent Triton™X-100 and forms an oligomeric structure estimated to be between dimer and tetramer but exhibits no apparent inducible structural dynamics neither chaperon-like activity in all the assays and molar ratios tested. SEC/FPLC and thermal aggregation prevention assays results indicate no formation of hetero-oligomeric complex or functional interactions between both sHSPs. Taken together these in vitro results portray the phage HspSP-ShM2 as a classical sHSP and suggest that it may be functional at the in vivo level while behaving differently than its host amphitropic sHSP.
我们之前报道了聚球藻噬菌体18 kDa小热休克蛋白（HspSP-ShM2）的计算机模拟特征。这种小热休克蛋白（sHSP）包含一个由92个氨基酸组成的高度保守的核心α晶体结构域以及相对较短的N端和C端臂，后者包含经典的C端锚定模块基序（L-X-I/L/V）。在此，我们利用尺寸排阻色谱法（SEC/FPLC）、梯度非变性凝胶电泳和动态光散射（DLS），在体外实验条件下确定了HspSP-ShM2的寡聚体概况及其结构动力学。在天然条件下，HspSP-ShM2表现出形成大寡聚体的能力，并呈现出多分散的分布。在较高温度下，它表现出广泛的结构动力学，并通过亚基重排增加和亚寡聚体物种的形成而发生构象变化。我们还证明了它在热休克条件下通过形成稳定且可溶的异源寡聚体复合物（sHSP:底物）来防止柠檬酸合酶、苹果酸脱氢酶和荧光素酶聚集的能力。相比之下，宿主蓝细菌聚球藻属WH7803的15 kDa sHSP（HspS-WH7803）在与HspSP-ShM2相同的条件下会聚集。然而，在非离子去污剂Triton™X-100存在的情况下，其溶解性可以得到维持，并形成一种估计介于二聚体和四聚体之间的寡聚结构，但在所有测试的测定和摩尔比中均未表现出明显的可诱导结构动力学或类似伴侣的活性。SEC/FPLC和热聚集预防测定结果表明，两种sHSP之间没有形成异源寡聚体复合物或功能相互作用。综合这些体外实验结果，表明噬菌体HspSP-ShM2是一种典型的sHSP，并表明它在体内水平可能具有功能，但其行为与宿主兼性sHSP不同。
