Kotamarthi Hema Chandra, Narayan Satya, Ainavarapu Sri Rama Koti
Department of Chemical Sciences, Tata Institute of Fundamental Research , Homi Bhabha Road, Colaba, Mumbai 400005, India.
J Phys Chem B. 2014 Oct 2;118(39):11449-54. doi: 10.1021/jp507463q. Epub 2014 Sep 19.
Folding and unfolding studies on large, multidomain proteins are still rare despite their high abundance in genomes of prokaryotes and eukaryotes. Here, we investigate the unfolding properties of a 271 residue, two-domain ribose binding protein (RBP) from the bacterial periplasm using single-molecule force spectroscopy. We observe that RBP predominately unfolds via a two-state pathway with an unfolding force of ∼80 pN and an unfolding contour length of ∼95 nm. Only a small population (∼15%) of RBP follows three-state pathways. The ligand binding neither increases the mechanical stability nor influences the unfolding flux of RBP through different pathways. The kinetic partitioning between two-state and three-state pathways, which has been reported earlier for other periplasmic proteins, is also observed in RBP, albeit to a lesser extent. These results provide important insights into the mechanical stability and unfolding processes of large two-domain proteins and highlight the contrasting features upon ligand binding. Protein structural topology diagrams are used to explain the differences in the mechanical unfolding behavior of RBP with other periplasmic binding proteins.
尽管大型多结构域蛋白在原核生物和真核生物基因组中大量存在，但对其进行折叠和去折叠研究的仍很少见。在此，我们使用单分子力谱技术研究了一种来自细菌周质的含271个残基的双结构域核糖结合蛋白（RBP）的去折叠特性。我们观察到，RBP主要通过双态途径去折叠，去折叠力约为80 pN，去折叠轮廓长度约为95 nm。只有一小部分（约15%）的RBP遵循三态途径。配体结合既不增加RBP的机械稳定性，也不影响其通过不同途径的去折叠通量。此前在其他周质蛋白中报道的双态和三态途径之间的动力学分配，在RBP中也有观察到，尽管程度较小。这些结果为大型双结构域蛋白的机械稳定性和去折叠过程提供了重要见解，并突出了配体结合后的对比特征。蛋白质结构拓扑图用于解释RBP与其他周质结合蛋白在机械去折叠行为上的差异。
