Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy
Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy
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DOI：
10.1007/s00775-015-1244-8
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发表时间：
2015-04-01
影响因子：
3
通讯作者：
Crowder, Michael W.
中科院分区：
文献类型：
--
作者：
Aitha, Mahesh;Moritz, Lindsay;Crowder, Michael W.
先前的晶体学和诱变研究已经暗示了金属β-内酰胺酶中位置保守的发夹环在底物结合和催化中的作用。为了探测该环在催化过程中的运动，使用快速冷冻淬灭双电子-电子共振（RFQ-DEER）光谱来询问金属-β-内酰胺酶CcrA，其在催化过程中在环上的位置49处具有自旋标记，并且在远离残基49的20-35处具有自旋标记（在位置82、126或233处）。在混合后10 ms，DEER谱显示位置49处的自旋标记与位置82、126和233处的自旋标记之间的距离分别增加7、10和13。与以前的假设相反，这些数据表明，在催化过程中，环移动近10远离金属中心，并且在催化过程中，环不夹紧在底物上。这项研究表明，催化过程中的环路运动可以在毫秒的时间尺度上进行查询。
Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-beta-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-beta-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.