Department of Molecular Physics, Huygens Laboratory, Leiden University, 2300, RA Leiden, The Netherlands.
Biopolymers. 2014 May;102(3):244-51. doi: 10.1002/bip.22467.
For 3D-structure determination in biophysical systems EPR is rapidly gaining ground. Proteins labeled specifically with two nitroxide spin labels can be prepared, and several EPR methods are available for distance determination, which makes it possible to determine distance constraints. However, such methods require frozen solutions, potentially causing non-physiological states of the sample. Here, we target spin- spin interaction in liquid solution at room temperature using rigid model compounds. A series of 310 -helical peptides, based on α-aminoisobutyric acid (Aib), is synthesized with pairs of spin labels separated by three, four, and five amino acids. To avoid flexibility, the noncoded nitroxyl-containing α-amino acid TOAC that is rigidly connected with the peptide backbone, is used. The EPR spectra of the peptides show a decreasing amount of coupling between the two spin labels within this series. We suggest through-bond interaction as the dominating mechanism for exchange interaction (J) and find a stronger J-coupling than in the corresponding Ala-based TOAC-peptides investigated previously (Hanson, et al., J Am Chem Soc 1996, 118, 7618-7625). We speculate that stronger coupling in Aib- vs Ala- peptides is due to intrinsically stronger through-bond interaction in the Aib-based peptides.
在生物物理系统的三维结构测定中，EPR 技术正在迅速发展。可以专门用两个氮氧自由基自旋标记物标记蛋白质，并可采用几种 EPR 方法进行距离测定，从而确定距离约束。然而，这些方法需要冷冻溶液，这可能导致样品处于非生理状态。在这里，我们在室温下使用刚性模型化合物来靶向液相中的自旋-自旋相互作用。基于α-氨基异丁酸（Aib）的一系列 310 螺旋肽被合成，其自旋标记物通过三个、四个和五个氨基酸隔开。为了避免灵活性，使用与肽骨架刚性连接的非编码含硝氧基的α-氨基酸 TOAC。肽的 EPR 光谱显示，在该系列中，两个自旋标记之间的耦合量逐渐减少。我们通过键间相互作用提出了交换相互作用（J）的主导机制，并发现了比以前研究过的基于 Ala 的 TOAC-肽更强的 J 耦合（Hanson 等人，J Am Chem Soc 1996，118，7618-7625）。我们推测，Aib-肽中更强的耦合是由于 Aib 基肽中固有更强的键间相互作用。
