Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Molecules. 2024 Jun 21;29(13):2950. doi: 10.3390/molecules29132950.
Long-range HNCO NMR spectra for proteins show crosspeaks due to J, J, J, and J couplings. The J couplings are transmitted through hydrogen bonds and their sizes are correlated to hydrogen bond lengths. We collected long-range HNCO data at a series of temperatures for four protein structures. P22i and CUS-3i are six-stranded beta-barrel I-domains from phages P22 and CUS-3 that share less than 40% sequence identity. The and states of the C-terminal domain from pore-forming toxin hemolysin ΙΙ (HlyIIC) arise from the isomerization of a single G404-P405 peptide bond. For P22i and CUS-3i, hydrogen bonds detected by NMR agree with those observed in the corresponding domains from cryoEM structures of the two phages. Hydrogen bond lengths derived from the J couplings, however, are poorly conserved between the distantly related CUS-3i and P22i domains and show differences even between the closely related and state structures of HlyIIC. This is consistent with hydrogen bond lengths being determined by local differences in structure rather than the overall folding topology. With increasing temperature, hydrogen bonds typically show an apparent increase in length that has been attributed to protein thermal expansion. Some hydrogen bonds are invariant with temperature, however, while others show apparent decreases in length, suggesting they become stabilized with increasing temperature. Considering the data for the three proteins in this study and previously published data for ubiquitin and GB3, lowered protein folding stability and cooperativity corresponds with a larger range of temperature responses for hydrogen bonds. This suggests a partial uncoupling of hydrogen bond energetics from global unfolding cooperativity as protein stability decreases.
长程 HNCO NMR 谱可用于显示蛋白质中的交叉峰，这些交叉峰归因于 J、J、J 和 J 偶合。这些 J 偶合通过氢键传递，其大小与氢键长度相关。我们在一系列温度下收集了四个蛋白质结构的长程 HNCO 数据。P22i 和 CUS-3i 是来自噬菌体 P22 和 CUS-3 的六股β桶 I 结构域，它们的序列同一性小于 40%。来自孔形成毒素溶细胞素 ΙΙ（HlyIIC）的 C 端结构域的 和 状态来自单个 G404-P405 肽键的异构化。对于 P22i 和 CUS-3i，NMR 检测到的氢键与两个噬菌体的相应结构域的冷冻电镜结构中观察到的氢键一致。然而，源自 J 偶合的氢键长度在远相关的 CUS-3i 和 P22i 结构域之间没有得到很好的保守，甚至在密切相关的 HlyIIC 的 和 状态结构之间也存在差异。这与氢键长度由局部结构差异而不是整体折叠拓扑决定是一致的。随着温度的升高，氢键通常表现出明显的长度增加，这归因于蛋白质的热膨胀。然而，有些氢键在温度上是不变的，而有些氢键则表现出明显的长度减小，表明它们随着温度的升高而变得更加稳定。考虑到本研究中三种蛋白质的数据以及先前发表的关于泛素和 GB3 的数据，降低的蛋白质折叠稳定性和协同性对应于氢键的温度响应范围更大。这表明随着蛋白质稳定性的降低，氢键的能量与整体展开协同性部分解耦。
