Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, Guangdong Province, China.
X-ray Science Division, Argonne National Laboratory, Argonne, IL, United States of America.
PLoS One. 2022 Apr 21;17(4):e0263102. doi: 10.1371/journal.pone.0263102. eCollection 2022.
Glutamine binding protein (GlnBP) is an Escherichia Coli periplasmic binding protein, which binds and carries glutamine to the inner membrane ATP-binding cassette (ABC) transporter. GlnBP binds the ligand with affinity around 0.1μM measured by isothermal titration calorimetry (ITC) and ligand binding stabilizes protein structure shown by its increase in thermodynamic stability. However, the molecular determinant of GlnBP ligand binding is not known. Electrostatic and hydrophobic interaction between GlnBP and glutamine are critical factors. We propose that the freedome of closure movement is also vital for ligand binding. In order to approve this hypothesis, we generate a series of mutants with different linker length that has different magnitude of domain closure. Mutants show different ligand binding affinity, which indicates that the propensity of domain closure determines the ligand binding affinity. Ligand binding triggers gradual ensemble conformational change. Structural changes upon ligand binding are monitored by combination of small angle x-ray scattering (SAXS) and NMR spectroscopy. Detailed structure characterization of GlnBP contributes to a better understanding of ligand binding and provides the structural basis for biosensor design.
谷氨酰胺结合蛋白（GlnBP）是一种大肠杆菌周质结合蛋白，它将谷氨酰胺结合并输送到内膜 ATP 结合盒（ABC）转运体。通过等温滴定量热法（ITC）测量，GlnBP 与配体的亲和力约为 0.1μM，配体结合稳定了蛋白质结构，表现为其热力学稳定性增加。然而，GlnBP 配体结合的分子决定因素尚不清楚。GlnBP 与谷氨酰胺之间的静电和疏水相互作用是关键因素。我们提出，封闭运动的自由度对于配体结合也是至关重要的。为了验证这一假设，我们生成了一系列具有不同连接长度的突变体，其结构域的封闭程度也不同。突变体显示出不同的配体结合亲和力，这表明结构域封闭的倾向决定了配体结合的亲和力。配体结合引发逐渐的整体构象变化。通过小角 X 射线散射（SAXS）和 NMR 光谱的结合监测配体结合引起的结构变化。GlnBP 的详细结构特征有助于更好地理解配体结合，并为生物传感器设计提供结构基础。
