W-Band pulse EPR distance measurements in peptides using Gd3+-dipicolinic acid derivatives as spin labels
W-Band pulse EPR distance measurements in peptides using Gd3+-dipicolinic acid derivatives as spin labels
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DOI：
10.1039/c1cp00011j
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发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Goldfarb, Daniella
中科院分区：
文献类型：
--
作者：
Gordon-Grossman, Michal;Kaminker, Ilia;Goldfarb, Daniella
我们提出了高场DEER（双电子-电子共振）距离测量使用Gd 3+（S = 7/2）自旋标记探测肽的构象在溶液中。使用Gd 3+自旋标记物作为标准氮氧自由基自旋标记物的替代物的动机是它们提供的灵敏度改进，因为它们在高磁场下具有非常强的EPR信号。Gd 3+通过吡啶二羧酸衍生物（4 MMDPA）标记配位，所述标记共价连接到两个半胱氨酸硫醇基团。在肽蜂毒肽的15和27位引入半胱氨酸，然后制备两种类型的自旋标记蜂毒肽，一种用两个氮氧自旋标记物标记，另一种用两个4 MMDPA-Gd 3+标记物标记。这两种类型都进行了W波段（95 GHz，3.5 T）的DEER测量。对于Gd 3+标记的肽，我们探讨了Gd 3+和标记肽的溶液摩尔比，温度和最大偶极演化时间T对DEER调制深度的影响。我们发现，[Gd 3 +]/[Tag]比例的优化是至关重要的，因为过量的Gd 3+掩盖了DEER效应，而太少的Gd 3+导致Gd 3 +-tag 2复合物的形成，产生肽二聚体。此外，我们观察到，DEER调制深度是敏感的光谱扩散过程，即使在Gd 3+浓度低至0.2 mM，因此实验条件应选择，以尽量减少它，因为它降低了DEER效应。最后，发现两个Gd 3+离子之间的距离为3.4 nm，比两个氮氧化物之间的距离长1.2 nm。这种差异的起源和影响进行了讨论。
We present high field DEER (double electron-electron resonance) distance measurements using Gd3+ (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd3+ spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd3+ was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd3+ labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd3+ labeled peptide we explored the effect of the solution molar ratio of Gd3+ and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd3+]/[Tag] ratio is crucial because excess Gd3+ masked the DEER effect and too little Gd3+ resulted in the formation of Gd3+-tag2 complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd3+ concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd3+ ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.