H. H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, United Kingdom.
Microsc Res Tech. 2011 Feb;74(2):170-6. doi: 10.1002/jemt.20888.
This study explores the mechanical unfolding of elastic protein analogues as a function of temperature, in both H₂O and D₂O, using atomic force microscopy (AFM) force spectroscopy in a specially constructed AFM liquid cell. This represents the first time that the effect of D₂O on protein flexibility has been investigated at the single molecule level by this technique. Model elastic peptides, R6, were encoded from synthetic genes expressed in Escherichia coli. The peptides possess short N- and C-terminal domains with central repetitive domains containing 13 repeats of the motif PGQGQQ-plus-GYYPTSLQQ. These sequences mimic those in native high molecular weight subunit glutenin proteins which confer elasticity to bread dough. Fitting single molecule stretching events to the worm-like chain model, allows determination of the molecular persistence length under various experimental conditions. The effect of increasing the temperature is to increase the persistence length of the peptide in both H₂O and D₂O, consistent with the expected "thermal softening" effect. However, the effect is significantly enhanced in D₂O, in which the persistence length at 45°C is ∼25% greater than the value measured in H₂O at the same temperature. Stronger intrapeptide H-bonding due to isotopic substitution of hydrogen for deuterium is the most likely cause of the enhanced backbone rigidity.
本研究探讨了弹性蛋白类似物在 H₂O 和 D₂O 中的机械展开作为温度的函数，使用原子力显微镜 (AFM) 力谱学在专门构建的 AFM 液体池中进行。这代表了首次通过该技术在单分子水平上研究 D₂O 对蛋白质柔韧性的影响。模型弹性肽 R6 由在大肠杆菌中表达的合成基因编码。这些肽具有短的 N-和 C-末端结构域，其中包含 13 个重复基序 PGQGQQ-plus-GYYPTSLQQ 的中心重复结构域。这些序列模拟了赋予面包面团弹性的天然高分子量亚基谷蛋白中的那些序列。将单分子拉伸事件拟合到蠕虫状链模型中，可以确定各种实验条件下的分子持久长度。增加温度的影响是增加肽在 H₂O 和 D₂O 中的持久长度，这与预期的“热软化”效应一致。然而，在 D₂O 中的影响要显著增强，在 45°C 时的持久长度比在相同温度下在 H₂O 中测量的值大 25%。由于氢被氘取代导致的更强的肽内氢键是增强主链刚性的最可能原因。
