Departments of Biological Sciences and Bioengineering, University of Texas at Dallas, Richardson, Texas75080, United States.
Department of Physics and Astronomy, Clemson University, Clemson, South Carolina29634, United States.
J Phys Chem B. 2023 Feb 2;127(4):884-898. doi: 10.1021/acs.jpcb.2c06720. Epub 2023 Jan 24.
The structural flexibility of proteins is crucial for their functions. Many experimental and computational approaches can probe protein dynamics across a range of time and length-scales. Integrative approaches synthesize the complementary outputs of these techniques and provide a comprehensive view of the dynamic conformational space of proteins, including the functionally relevant limiting conformational states and transition pathways between them. Here, we introduce an integrative paradigm to model the conformational states of multidomain proteins. As a model system, we use the first two tandem PDZ domains of postsynaptic density protein 95. First, we utilize available sequence information collected from genomic databases to identify potential amino acid interactions in the PDZ1-2 tandem that underlie modeling of the functionally relevant conformations maintained through evolution. This was accomplished through combination of coarse-grained structural modeling with outputs from direct coupling analysis measuring amino acid coevolution, a hybrid approach called SBM+DCA. We recapitulated five distinct, experimentally derived PDZ1-2 tandem conformations. In addition, SBM+DCA unveiled an unidentified, twisted conformation of the PDZ1-2 tandem. Finally, we implemented an integrative framework for the design of single-molecule Förster resonance energy transfer (smFRET) experiments incorporating the outputs of SBM+DCA with simulated FRET observables. This resulting FRET network is designed to mutually resolve the predicted limiting state conformations through global analysis. Using simulated FRET observables, we demonstrate that structural modeling with the newly designed FRET network is expected to outperform a previously used empirical FRET network at resolving all states simultaneously. Integrative approaches to experimental design have the potential to provide a new level of detail in characterizing the evolutionarily conserved conformational landscapes of proteins, and thus new insights into functional relevance of protein dynamics in biological function.
蛋白质的结构灵活性对其功能至关重要。许多实验和计算方法可以在不同的时间和长度尺度上探测蛋白质的动力学。综合方法综合了这些技术的互补输出，提供了蛋白质动态构象空间的综合视图，包括功能相关的限制构象状态和它们之间的转换途径。在这里，我们介绍了一种综合范例来模拟多结构域蛋白质的构象状态。作为一个模型系统，我们使用突触后密度蛋白 95 的前两个串联 PDZ 结构域。首先，我们利用从基因组数据库中收集的可用序列信息，确定 PDZ1-2 串联中潜在的氨基酸相互作用，这些相互作用是通过进化保持功能相关构象的建模基础。这是通过粗粒度结构建模与直接耦合分析测量氨基酸共进化的输出相结合来实现的，这是一种称为 SBM+DCA 的混合方法。我们重现了五个独特的、实验衍生的 PDZ1-2 串联构象。此外，SBM+DCA 揭示了 PDZ1-2 串联的一个未识别的扭曲构象。最后，我们实现了一个综合框架，用于设计单分子荧光共振能量转移 (smFRET) 实验，将 SBM+DCA 的输出与模拟的 FRET 可观察值结合起来。这个得到的 FRET 网络旨在通过全局分析相互解析预测的限制状态构象。使用模拟的 FRET 可观察值，我们证明了使用新设计的 FRET 网络进行结构建模预计将比以前使用的经验 FRET 网络在同时解析所有状态方面表现更好。综合实验设计方法有可能提供一个新的细节水平，以描述蛋白质进化保守的构象景观，从而深入了解蛋白质动力学在生物学功能中的功能相关性。
