Savitsky A, Dubinskii A A, Plato M, Grishin Y A, Zimmermann H, Möbius K
Department of Physics, Free University Berlin, Arnimallee 14, D-14195 Berlin, Germany.
J Phys Chem B. 2008 Jul 31;112(30):9079-90. doi: 10.1021/jp711640p. Epub 2008 Jul 2.
The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.
高场电子顺磁共振（EPR）与采用氮氧化物自由基的定点自旋标记（SDSL）技术相结合，在揭示嵌入膜中的蛋白质极性和质子化特征的细微变化方面已被证明特别有效。该信息可通过取向选择性高场EPR获得，该方法可解析附着于特定分子位点的自旋标记的氮氧化物塞曼（g）张量和超精细（A）张量的主成分。与g张量和A张量不同，氮氧化物自旋标记的（14）N（I = 1）四极相互作用张量尚未在EPR中用于探测功能蛋白位点微环境的影响。在这项工作中表明，W波段（95 GHz）高场电子自旋回波包络调制（ESEEM）方法非常适合在无序冷冻溶液中高精度地确定氮氧化物自旋标记的（14）N四极张量主成分。通过W波段ESEEM，已确定了玻璃态邻三联苯和甘油溶液中五环吡咯啉型氮氧化物自由基的四极成分。该自由基是SDSL蛋白质研究中广泛使用的MTS自旋标记的头基。通过密度泛函理论计算和W波段ESEEM实验表明，在氢键和非键合情况下，Q（yy）值对氮氧化物环境的质子化和极性特别敏感。四极张量对氮氧化物标记本身的结构变化相当不敏感。当使用Q（yy）作为环境测试探针时，其对温度变化的稳定性相对于通常用于探测基质对自旋标记分子位点影响的g xx和A（zz）参数具有重要优势。因此，除了测量无序固体中自旋标记蛋白位点的g xx和A（zz）之外，对（14）N四极相互作用的W波段高场ESEEM研究为可靠地探测对电子结构的细微环境影响开辟了一条新途径。这是在区分基质极性和氢键形成效应方面向前迈出的重要一步。
