J Am Chem Soc. 2019 May 8;141(18):7320-7326. doi: 10.1021/jacs.9b00577. Epub 2019 Apr 24.
Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain C═O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain C═O. We use side chain C═O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the C═O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain C═O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies.
红外（IR）光谱学为淀粉样纤维的结构、动力学和形成机制提供了重要的见解。IR 探针，如主链 C=O，已被广泛用于获取特定结构的信息，但只能探测到二级结构和链到链的排列。很少有非干扰性的 IR 探针可用于报告侧链构象和环境，这对于确定淀粉样纤维中的层到层排列至关重要。极性残基，如谷氨酰胺，对淀粉样蛋白的稳定性有重要贡献，因此经常出现在淀粉样肽/蛋白的核心区域。此外，聚谷氨酰胺（polyQ）重复序列在几种神经退行性疾病中形成有毒聚集体。在这里，我们报告了一种新的非干扰性 IR 探针-谷氨酰胺侧链 C=O 的合成和应用。我们使用侧链 C=O 标记和同位素稀释来检测淀粉样形成肽中存在的分子间氢键排列的谷氨酰胺侧链（谷氨酰胺梯）。此外，C=O 峰的线宽对其局部水合环境非常敏感。侧链标记的 IR 数据使我们能够明确确定短淀粉样形成肽 GNNQQNY 的层到层排列，从而提供了通过主链标记无法获得的见解。使用几个不同的纤维样品，我们还展示了这种 IR 探针在研究淀粉样纤维的结构和聚集动力学方面的多功能性。最后，我们证明了使用集成建模平台（IMP）和将 IR 与其他生物物理方法结合使用以进行更准确的结构建模的能力，IR 数据在建模淀粉样结构方面的能力。总的来说，我们相信侧链 C=O 将在未来的淀粉样体 IR 研究中补充主链同位素标记，并且使用 IR 数据进行的集成建模将显著扩展 IR 光谱在阐明淀粉样体组装方面的作用。
