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Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity.
International Journal of Biological Macromolecules ( IF 8.2 ) Pub Date : 2020-09-19 , DOI: 10.1016/j.ijbiomac.2020.09.095
Yi-Jun Shi , Jing-Ting Chiou , Chia-Hui Huang , Yuan-Chin Lee , Liang-Jun Wang , Long-Sen Chang

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.



中文翻译:

羧基的修饰将α-乳清蛋白转化为具有膜扰动活性和细胞毒性的活性熔融小球状态。

我们研究了用氨基脲对带负电荷的羧基进行修饰是否可以赋予牛α-乳清蛋白(LA)膜破坏性和细胞毒性。MALDI-TOF分析表明,洛杉矶的21个羧基中有18个与氨基脲分子偶联。圆二色性光谱的测量和Trp荧光猝灭研究表明,氨基脲修饰的LA(SEM-LA)具有熔融的球状构象,保留了α-螺旋二级结构,但失去了LA的三级结构。与LA相比,SEM-LA在响应三氟乙醇和温度诱导的结构转变时具有更高的结构柔性。与LA形成鲜明对比的是,SEM-LA表现出膜破坏活性和细胞毒性。此外,SEM-LA诱导的膜通透性促进了柔红霉素的吸收,从而促进了其细胞毒性。碘化物猝灭研究表明,SEM-LA Trp残基周围的微环境富含正电荷。SEM-LA与脂质囊泡的结合改变了Trp残基周围带正电荷的簇。尽管LA和SEM-LA表现出相似的脂质结合亲和力,但SEM-LA和LA的膜相互作用模式却有所不同。总的来说,这些结果表明带负电荷的残基的阻断使得能够形成具有结构柔性和增加的正电荷的LA的熔融球构象,从而产生具有膜破坏活性和细胞毒性的功能性LA。碘化物猝灭研究表明,SEM-LA Trp残基周围的微环境富含正电荷。SEM-LA与脂质囊泡的结合改变了Trp残基周围带正电荷的簇。尽管LA和SEM-LA表现出相似的脂质结合亲和力,但SEM-LA和LA的膜相互作用模式却有所不同。总的来说,这些结果表明带负电荷的残基的阻断使得能够形成具有结构柔性和增加的正电荷的LA的熔融球构象,从而产生具有膜破坏活性和细胞毒性的功能性LA。碘化物猝灭研究表明,SEM-LA Trp残基周围的微环境富含正电荷。SEM-LA与脂质囊泡的结合改变了Trp残基周围带正电荷的簇。尽管LA和SEM-LA表现出相似的脂质结合亲和力,但SEM-LA和LA的膜相互作用模式却有所不同。总的来说,这些结果表明带负电荷的残基的阻断使得能够形成具有结构柔性和增加的正电荷的LA的熔融球构象,从而产生具有膜破坏活性和细胞毒性的功能性LA。尽管LA和SEM-LA表现出相似的脂质结合亲和力,但SEM-LA和LA的膜相互作用模式却有所不同。总的来说,这些结果表明带负电荷的残基的阻断使得能够形成具有结构柔性和增加的正电荷的LA的熔融球构象,从而产生具有膜破坏活性和细胞毒性的功能性LA。尽管LA和SEM-LA表现出相似的脂质结合亲和力,但SEM-LA和LA的膜相互作用模式却有所不同。总的来说,这些结果表明带负电荷的残基的阻断使得能够形成具有结构柔性和增加的正电荷的LA的熔融球构象,从而产生具有膜破坏活性和细胞毒性的功能性LA。

更新日期:2020-09-20
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