Department of Neuroscience, University of Wisconsin, Madison, Wisconsin.
School of Pharmacy, Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin.
Biophys J. 2013 Dec 17;105(12):2724-32. doi: 10.1016/j.bpj.2013.11.010.
Measurements of inter- and intramolecular distances are important for monitoring structural changes and understanding protein interaction networks. Fluorescence resonance energy transfer and functionalized chemical spacers are the two predominantly used strategies to map short-range distances in living cells. Here, we describe the development of a hybrid approach that combines the key advantages of spectroscopic and chemical methods to estimate dynamic distance information from labeled proteins. Bifunctional spectroscopic probes were designed to make use of adaptable-anchor and length-varied spacers to estimate molecular distances by exploiting short-range collisional electron transfer. The spacers were calibrated using labeled polyproline peptides of defined lengths and validated by molecular simulations. This approach was extended to estimate distance restraints that enable us to evaluate the resting-state model of the Shaker potassium channel.
分子间和分子内距离的测量对于监测结构变化和理解蛋白质相互作用网络非常重要。荧光共振能量转移和功能化化学间隔物是两种主要用于在活细胞中绘制短程距离的策略。在这里，我们描述了一种混合方法的发展，该方法结合了光谱和化学方法的关键优势，从标记的蛋白质中估计动态距离信息。双功能光谱探针的设计利用了适应性锚定和长度变化的间隔物，通过利用短程碰撞电子转移来估计分子距离。使用具有定义长度的标记多聚脯氨酸肽校准间隔物，并通过分子模拟进行验证。该方法扩展到估计距离约束，使我们能够评估 Shaker 钾通道的静止状态模型。
