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 LC-MS/MS. 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.

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使用PTAD与酪氨酸结合鉴别蛋白质结构的变化

蛋白质的化学修饰在工程蛋白质疗法，靶向生物药物，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，因此使其可以位于蛋白质表面或结合界面，或被埋在蛋白质中。酪氨酸可以与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。我们将酪氨酸残基缀合在折叠良好的蛋白质，牛血清白蛋白（BSA）中，并用LC-MS / MS定量标记的酪氨酸。我们将此方法应用于尿素存在下产生的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。这项研究证明了将PTAD用作酪氨酸结合的新用途，该分析工具能够区分蛋白质的构象状态。