Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation
Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation
复制标题
DOI：
10.1016/j.bbamem.2021.183680
复制
发表时间：
2021-06-29
影响因子：
3.4
通讯作者：
Yamazaki, Masahito
中科院分区：
文献类型：
--
作者：
Ali, Md Hazrat;Shuma, Madhabi Lata;Yamazaki, Masahito
荧光探针标记的多肽被用来研究多肽与细胞和脂泡的相互作用，但用荧光探针标记多肽可以显着改变这些相互作用。我们最近开发了一种新的方法来检测未标记的多肽进入单个巨大的单层囊泡(GUV)的管腔。在这里，我们应用这种方法来检测抗菌肽PGLa与单个GUVS的相互作用，以阐明PGLa是否能够进入GUVO腔而不形成孔。首先，我们研究了未标记的PGLa与由双油酰磷脂酰甘油(DOPG)和双油酰磷脂酰胆碱(DOPC)(4/6)组成的单层胶体之间的相互作用，这些胶体的管腔含有荧光探针AF647和DOPG/DOPC(8/2)-包裹着高浓度钙黄绿素的大单层囊泡。在开始与PGLa相互作用一段较长的滞后期后，钙黄素(Icalcein)引起的GUV管腔的荧光强度逐渐增加，而AF647没有渗漏，这表明PGLa进入GV管腔时没有在GV膜上形成孔洞。随着PGLa浓度的增加，PGLa的进入比例增加。同时测定GUV膜面积变化分数(D)和PGLa引起的Icalcein增加，表明PGLa仅在d第二次增加时才进入管腔，表明PGLa是在从外叶向内叶转位的过程中进入管腔的。随着膜张力的增加，未形成孔的PGLa进入膜的比例增加。在此基础上，我们讨论了PGLa进入胃腔的基本过程和机制。
Fluorescent-probe-labeled peptides are used to study the interactions of peptides with cells and lipid vesicles but labeling peptides with fluorescent probes can significantly change these interactions. We recently developed a new method to detect the entry of nonlabeled peptides into the lumen of single giant unilamellar vesicles (GUVs). Here we applied this method to examine the interaction of the antimicrobial peptide PGLa with single GUVs to elucidate whether PGLa can enter the GUV lumen without pore formation. First, we examined the interaction of nonlabeled PGLa with single GUVs comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6) whose lumens contain the fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles encapsulating a high concentration of calcein. After a large lag period from starting the interaction with PGLa, the fluorescence intensity of the GUV lumen due to calcein (Icalcein) increased gradually without leakage of AF647, indicating that PGLa enters the GUV lumen without pore formation in the GUV membrane. The fraction of entry of PGLa increased with increasing PGLa concentration. Simultaneous measurement of the fractional area change of the GUV membrane (d) and PGLa-induced increase in Icalcein showed that the entry of PGLa occurs only during the second increase in d, indicating that PGLa enters the lumen during its translocation from the outer leaflet to the inner leaflet. The fraction of entry of PGLa without pore formation increased with increasing membrane tension. Based on these results, we discuss the elementary processes and the mechanism of the entry of PGLa into the GUV lumen.