Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada.
J Am Soc Mass Spectrom. 2013 Jun;24(6):907-16. doi: 10.1007/s13361-013-0599-8. Epub 2013 Apr 18.
The effects of binding two small-molecule inhibitors to Agrobacterium sp. strain ATCC 21400 (Abg) β-glucosidase on the conformations and stability of gas-phase ions of Abg have been investigated. Biotin-iminosugar conjugate (BIC) binds noncovalently to Abg while 2,4-dinitro-2-deoxy-2-fluoro-β-D-glucopyranoside (2FG-DNP) binds covalently with loss of DNP. In solution, Abg is a dimer. Mass spectra show predominantly dimer ions, provided care is taken to avoid dissociation of dimers in solution and dimer ions in the ion sampling interface. When excess inhibitor, either covalent or noncovalent, is added to solutions of Abg, mass spectra show peaks almost entirely from 2:2 inhibitor-enzyme dimer complexes. Tandem mass spectrometry experiments show similar dissociation channels for the apo-enzyme and 2FG-enzyme dimers. The +21 dimer produces +10 and +11 monomers. The internal energy required to dissociate the +21 2FG-enzyme to its monomers (767 ± 30 eV) is about 36 eV higher than that for the apo-enzyme dimer (731 ± 6 eV), reflecting the stabilization of the free enzyme dimer by the 2FG inhibitor. The primary dissociation channels for the noncovalent BIC-enzyme dimer are loss of neutral and charged BIC. The internal energy required to induce loss of BIC is 482 ± 8 eV, considerably less than that required to dissociate the dimers. For a given charge state, ions of the covalent and noncovalent complexes have about 15 % and 25 % lower cross sections, respectively, compared with the apo-enzyme. Thus, binding the inhibitors causes the gas-phase protein to adopt more compact conformations. Noncovalent binding surprisingly produces the greatest change in protein ion conformation, despite the weaker inhibitor binding. ᅟ
已经研究了将两种小分子抑制剂与根癌农杆菌（Agrobacterium sp.）菌株 ATCC 21400（Abg）β-葡萄糖苷酶结合对 Abg 气相离子构象和稳定性的影响。生物素亚氨基糖缀合物（BIC）非共价结合 Abg，而 2,4-二硝基-2-脱氧-2-氟-β-D-吡喃葡萄糖（2FG-DNP）与 Abg 共价结合，同时失去 DNP。在溶液中，Abg 是二聚体。质谱显示主要是二聚体离子，只要注意避免在溶液中解离二聚体和在离子采样界面中的二聚体离子。当向 Abg 溶液中加入过量的抑制剂（无论是共价的还是非共价的）时，质谱显示几乎完全来自 2:2 抑制剂-酶二聚体复合物的峰。串联质谱实验显示，apo-酶和 2FG-酶二聚体的解离通道相似。+21 二聚体产生+10 和+11 单体。将+21 2FG-酶解聚为其单体所需的内部能量（767±30 eV）比 apo-酶二聚体（731±6 eV）高约 36 eV，这反映了 2FG 抑制剂对游离酶二聚体的稳定作用。非共价 BIC-酶二聚体的主要解离通道是中性和带电 BIC 的丢失。诱导 BIC 丢失所需的内部能量为 482±8 eV，远低于二聚体的解离所需能量。对于给定的电荷状态，与 apo-酶相比，共价和非共价复合物的离子的截面分别低约 15%和 25%。因此，结合抑制剂会使气相蛋白采用更紧凑的构象。尽管结合较弱，但令人惊讶的是，非共价结合会导致蛋白质离子构象发生最大变化。
