PDF(Pubmed)
Abstract：
Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1H-15N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation.
摘要：
脱酰胺经常被称为晶状体蛋白聚集和白内障形成的重要驱动因素。这里,我们表征了γD-晶状体蛋白中Asn对Asp的累积变化的结构和生物物理后果。使用NMR光谱，我们证明了N-或C-末端结构域限制或完全Asn到Asp改变的γD-晶状体蛋白表现出与野生型蛋白基本相同的1H-15NHSQC谱，这意味着保留了整体结构。通过离液展开观察到整个Asn到AspγD-晶状体蛋白变体只有很小的热力学不稳定，以及胶体稳定性的评估，通过测量扩散相互作用参数，在关联倾向上没有实质性差异。此外，使用分子动力学模拟，未检测到Asn到Asp或iso-Asp变化的蛋白质动力学的显着变化。我们的综合结果表明，所有Asn被Asp残基取代，反映了脱酰胺的极端情况，不影响γD-晶状体蛋白的结构和生物物理特性。这表明这些变化本身不能成为驱动白内障形成的主要决定因素。
