DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.
DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.
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DOI：
10.1021/acs.jpclett.6b00456
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发表时间：
2016-04-21
期刊：
影响因子：
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通讯作者：
Smith GM
中科院分区：
文献类型：
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作者：
Motion CL;Lovett JE;Bell S;Cassidy SL;Cruickshank PA;Bolton DR;Hunter RI;El Mkami H;Van Doorslaer S;Smith GM
这项工作表明，在94 GHz的双电子-电子共振（DEER）脉冲EPR技术的可行性，使灵敏的铁（III）血红素中心和氮氧自由基自旋标记蛋白质之间的纳米距离测量。使用DEER精确测量许多类别血红素蛋白长距离的技术目前受到灵敏度的强烈限制。在本文中，我们证明了超过30倍的灵敏度增益与以前的较低频率（X波段）DEER测量人类脑红蛋白和抹香鲸肌红蛋白。这是通过利用最新的仪器进步，采用宽带激发技术的基础上复合脉冲和利用更有利的弛豫性能的低自旋Fe（III）在高磁场。这种灵敏度的提高可能使DEER技术被常规地用作大量血红素和许多其他金属蛋白的结构和构象的灵敏探针。
This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.