The development of chemical strategies for site-specific protein modification now enables researchers to attach polyethylene glycol (PEG) to a protein drug at one or more specific locations (i.e., protein PEGylation). However, aside from avoiding enzyme active sites or protein-binding interfaces, distinguishing the optimal PEGylation site from the available alternatives has conventionally been a matter of trial and error. As part of a continuing effort to develop guidelines for identifying optimal PEGylation sites within proteins, we show here that the impact of PEGylation at various sites within the β-sheet model protein WW depends strongly on the identity of the PEG–protein linker. The PEGylation of Gln or of azidohomoalanine has a similar impact on WW conformational stability as does Asn–PEGylation, whereas the PEGylation of propargyloxyphenylalanine is substantially stabilizing at locations where Asn–PEGylation was destabilizing. Importantly, we find that at least one of these three site-specific PEGylation strategies leads to substantial PEG-based stabilization at each of the positions investigated, highlighting the importance of considering conjugation strategy as an important variable in selecting optimal PEGylation sites. We further demonstrate that using a branched PEG oligomer intensifies the impact of PEGylation on WW conformational stability and also show that PEG-based increases to conformational stability are strongly associated with corresponding increases in proteolytic stability.

中文翻译：

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基于聚乙二醇的β-Sheet蛋白构象和蛋白水解稳定性变化取决于共轭策略和位置

现在，针对位点特异性蛋白质修饰的化学策略的发展使研究人员能够在一个或多个特定位置（即蛋白质PEG化）将聚乙二醇（PEG）连接到蛋白质药物上。然而，除了避免酶活性位点或蛋白质结合界面之外，将最佳的PEG化位点与可用的替代物区分开通常是一个反复试验的问题。作为制定指导原则以鉴定蛋白质中最佳PEG化位点的持续努力的一部分，我们在这里表明PEG化对β-sheet模型蛋白质WW内各个位点的影响在很大程度上取决于PEG-蛋白质接头的身份。Gln或叠氮高丙氨酸的PEG化对WW构象稳定性的影响与Asn–PEG化类似，而炔丙氧基苯丙氨酸的PEG化在Asn-PEG化不稳定的位置基本稳定。重要的是，我们发现这三种位点特异性PEG化策略中的至少一种在每个研究的位置上都导致了基于PEG的基本稳定，突出了将缀合策略视为选择最佳PEG化位点的重要变量的重要性。我们进一步证明，使用支链PEG低聚物可增强PEG化对WW构象稳定性的影响，并且还表明基于PEG的构象稳定性增加与相应的蛋白水解稳定性增加密切相关。我们发现这三种位点特异性PEG化策略中的至少一种在每个研究的位置上都导致了基于PEG的稳定，这突出了考虑将缀合策略作为选择最佳PEG化位点的重要变量的重要性。我们进一步证明，使用支链PEG低聚物可增强PEG化对WW构象稳定性的影响，并且还表明基于PEG的构象稳定性增加与相应的蛋白水解稳定性增加密切相关。我们发现这三种位点特异性PEG化策略中的至少一种在每个研究的位置上都导致了基于PEG的稳定，这突出了考虑将缀合策略作为选择最佳PEG化位点的重要变量的重要性。我们进一步证明，使用支链PEG低聚物可增强PEG化对WW构象稳定性的影响，并且还表明基于PEG的构象稳定性增加与相应的蛋白水解稳定性增加密切相关。