Chemical modification of proteins has been crucial in engineering protein‐based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation‐based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4‐phenyl‐3H‐1,2,4‐triazole‐3,5(4H)‐dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well‐folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with liquid chromatography with tandem mass spectrometry. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein.

中文翻译：

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使用酪氨酸共轭区分蛋白质结构的变化。

蛋白质的化学修饰在工程化基于蛋白质的疗法、靶向生物药剂学、分子探针和生物材料中至关重要。在这里，我们探索使用基于共轭的方法来感知蛋白质中的替代构象状态。酪氨酸具有疏水性和亲水性，因此可以定位在蛋白质表面，或结合界面，或被埋在蛋白质中。酪氨酸可与 4-苯基-3H-1,2,4-三唑-3,5(4H)-二酮 (PTAD) 结合。我们假设可以通过用 PTAD 标记蛋白质中的酪氨酸残基来区分单个蛋白质构象。我们结合了折叠良好的蛋白质牛血清白蛋白 (BSA) 中的酪氨酸残基，并通过液相色谱和串联质谱法对标记的酪氨酸进行了定量。我们将这种方法应用于在尿素存在下产生的 BSA 的替代构象。发现 PTAD 标记的量与每个酪氨酸相对于蛋白质表面的深度有关。这项研究证明了酪氨酸缀合的新用途，它使用 PTAD 作为能够区分蛋白质构象状态的分析工具。