Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan.
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan; Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.
Arch Biochem Biophys. 2022 Oct 15;728:109371. doi: 10.1016/j.abb.2022.109371. Epub 2022 Jul 31.
In this study, we investigated whether modification of the carboxyl group with semicarbazide-enabled myoglobin (Mb) exhibits membrane-perturbing activity in physiological solutions. Mass spectrometry analysis showed that semicarbazide molecules were coupled to 19 of the 22 carboxyl groups in semicarbazide-modified Mb (SEM-Mb). Measurements of the absorption and circular dichroism spectra indicated that SEM-Mb lost its heme group and reduced the content of the α-helix structure in Mb. The microenvironment surrounding Trp residues in Mb changes after blocking negatively charged residues, as shown by fluorescence quenching studies. The results of the trifluoroethanol-induced structural transition indicated that SEM-Mb had higher structural flexibility than that of Mb. SEM-Mb, but not Mb, induced the permeability of bilayer membranes. Both proteins showed similar lipid-binding affinities. The conformation of SEM-Mb and Mb changed upon binding to lipid vesicles or a membrane-mimicking environment composed of SDS micelles, suggesting that membrane interaction modes differ. Unlike lipid-bound Mb, Trp residues in lipid-bound SEM-Mb are located at the protein-lipid interface. Altogether, our data indicate that modifying negatively charged groups relieves the structural constraints in Mb, consequently switching Mb structure to an active conformation that exhibits membrane-permeabilizing activity.
在这项研究中，我们研究了羧基的半缩脲化修饰是否会使肌红蛋白（Mb）在生理溶液中表现出膜扰动活性。质谱分析表明，半缩脲分子与半缩脲修饰肌红蛋白（SEM-Mb）中的 22 个羧基中的 19 个发生了偶联。吸收和圆二色性光谱的测量表明，SEM-Mb 失去了其血红素基团，并降低了 Mb 中α-螺旋结构的含量。荧光猝灭研究表明，在封闭带负电荷的残基后，Mb 中色氨酸残基周围的微环境发生了变化。三氟乙醇诱导的结构转变的结果表明，SEM-Mb 比 Mb 具有更高的结构灵活性。SEM-Mb 而不是 Mb 诱导双层膜的通透性。两种蛋白质均显示出相似的脂质结合亲和力。SEM-Mb 和 Mb 与脂质囊泡或由 SDS 胶束组成的膜模拟环境结合后，构象发生变化，表明膜相互作用模式不同。与脂质结合的 Mb 不同，脂质结合的 SEM-Mb 中的色氨酸残基位于蛋白质-脂质界面。总之，我们的数据表明，修饰带负电荷的基团可以减轻 Mb 中的结构约束，从而将 Mb 结构转换为具有膜通透活性的活性构象。
