Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13 C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the “proton-less” nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

中文翻译：

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HIF-1α 固有无序区域的脯氨酰羟基化的时间分辨 NMR 检测


脯氨酰羟基化是一种氧依赖性翻译后修饰 （PTM），已知可调节胶原蛋白的原纤维形成并调节缺氧诱导因子 （HIF） α亚基的细胞表达。然而，由于缺乏可以直接测量固有无序蛋白中多种脯氨酰羟基化事件的生物物理方法，我们对这种重要但相对罕见的 PTM 的理解仍然不完整。在这里，我们描述了一种基于实时 13 C 直接检测 NMR 的分析方法，用于通过利用脯氨酸的“无质子”性质，在缺氧诱导因子 1α 的固有无序氧依赖性降解域中同时研究两种进化上保守的脯氨酸（P402 和 P564）的羟基化。我们明确表明 P564 以时间分辨方式快速羟基化，而 P402 羟基化明显滞后于 P564。差异羟基化速率受与脯氨酰羟化酶结合亲和力的影响可以忽略不计，而是受周围氨基酸组成的影响，特别是 P564 在 +1 位的保守酪氨酸残基。这些发现支持了一个出乎意料的观点，即进化上保守的 P402 似乎对正常的氧感应途径的影响很小。