Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
PLoS One. 2013 May 28;8(5):e64686. doi: 10.1371/journal.pone.0064686. Print 2013.
The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.
1 型兰尼碱受体（RyR1）是一种细胞内 Ca²⁺释放通道，介导骨骼肌兴奋收缩耦联。虽然使用冷冻电子显微镜重建已经阐明了 RyR1 的整体形状，但精细的结构细节仍然难以捉摸。为了更好地了解 RyR1 的结构，我们之前使用了一种基于细胞的荧光共振能量转移（FRET）方法，该方法使用融合的绿色荧光蛋白（GFP）供体和荧光受体 Cy3NTA，Cy3NTA 特异性结合工程到 RyR1 中的短多组氨酸“标签”。然而，需要透化细胞以允许 Cy3NTA 进入以及 Cy3NTA 与 His 标签的非共价结合限制了该技术在研究 RyR 构象变化中的未来应用。为了克服这些问题，我们使用包含四半胱氨酸（Tc）基序的十二肽序列将双砷荧光团 FlAsH 和 ReAsH 靶向到 RyR1。这些化合物自由穿过完整的细胞膜，然后在那里与 Tc 基序共价结合。首先，我们通过将黄色荧光蛋白（YFP）FRET 供体融合到 RyR1 的 N 端，然后将 FRET 受体 ReAsH 靶向到相邻的 Tc 标签，使用该系统在完整细胞中进行 FRET 测量。观察到中等能量转移（约 33%），而缺乏 Tc 标签的 YFPRyR1 融合蛋白孵育导致可检测的 FRET 没有。我们还开发了一种不需要 RyR 荧光蛋白融合的 FRET 系统，方法是用 FRET 供体 FlAsH 标记 N 端 Tc 标记的 RyR1，然后将 FRET 受体 Cy3NTA 靶向到相邻的十组氨酸（His10）标签。高度的能量转移（约 66%）表明两种化合物与 RyR1 中的这些独特识别序列的正确结合。因此，这两个系统应该为未来基于 FRET 的 RyR1 结构测定提供前所未有的灵活性。
