Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins

Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein–protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N3– (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C–H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana, we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an “affinity reagent” for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.疏水微区（hydrophobic microdomains），亦称疏水补丁（hydrophobic patches），对水溶性蛋白质的诸多重要生物学功能而言至关重要。其功能涵盖配体/底物结合、蛋白质-蛋白质相互作用、翻译后正确折叠以及变性过程中的聚集行为。与可通过蛋白质一级序列中连续的疏水氨基酸侧链区段轻松识别的跨膜结构域（transmembrane domains）不同，这类三维疏水补丁难以被精准预测。缺乏可直接确定其位置的实验策略，阻碍了学界对其结构与功能的进一步研究。本研究提出，小型三原子阴离子N3⁻（叠氮，azide）会被这类疏水补丁所吸引，在氧化剂存在的条件下形成自由基，对邻近氨基酸的C-H键进行共价修饰。本研究使用两种模型蛋白（BSA与溶菌酶）以及模式高等植物拟南芥（Arabidopsis thaliana）的无细胞裂解液开展实验，发现自由基介导的共价叠氮化修饰可发生在埋藏的催化活性位点与配体结合位点内，且其行为与已报道的疏水探针（hydrophobic probes）一致。本研究结果支持如下模型：叠氮自由基（azido radical）可作为非水相三维蛋白质微环境的亲和试剂（"affinity reagent"），这一结论与叠氮基团的非定域电子密度特性，以及有机合成中广泛使用的叠氮自由基已知的高反应活性均相符。本研究提出，叠氮自由基可作为一种简便的手段，用于鉴定水溶性蛋白质中的疏水微环境；此外，该方法无需基因操作或特殊试剂，即可为蛋白质连接化学标签，是一种全新的简便策略。