Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, 8380494, Chile.
Department of Physics and Astronomy, University of California - Los Angeles, Los Angeles, California, 90095, US.
Protein Sci. 2018 Aug;27(8):1418-1426. doi: 10.1002/pro.3432.
Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.
免疫球蛋白结合蛋白（BiP）是伴侣蛋白和分子马达，属于 Hsp70 家族，参与内质网中蛋白质合成、折叠和易位等重要生物学过程的调节。BiP 有两个高度保守的结构域：N 端核苷酸结合结构域（NBD）和 C 端底物结合结构域（SBD），由一个疏水性接头连接。ATP 在 NBD 中结合并水解为 ADP，BiP 的延伸多肽底物在 SBD 中结合。像许多分子马达一样，BiP 的功能取决于结构和催化特性，这些特性可能有助于其性能。一种研究 BiP 机械特性的新方法是通过纳米流变学技术研究配体结合时粘弹性行为的变化。该技术本质上是一种传统的流变学实验，在该实验中，直接向研究中的蛋白质施加振荡力，并测量产生的平均变形。我们的结果表明，蛋白质的折叠状态表现为粘弹性材料，在结合核苷酸-ATP、ADP 和 AMP-PNP 时变得更软，但在结合 HTFPAVL 肽底物时变得更硬。此外，我们观察到肽结合显著增加了对 ADP 的亲和力，使其解离常数（Kd）降低约 1000 倍，证明了 SBD 和 NBD 结构域之间的变构偶联。
