Gordon Emma A, Sahu Indra D, Fried Joel R, Lorigan Gary A
Materials Science and Technology Division, Los Alamos National Lab, Los Alamos, NM 87545, USA.
Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA.
Biomimetics (Basel). 2025 Mar 2;10(3):154. doi: 10.3390/biomimetics10030154.
Studying membrane proteins in a native environment is crucial to understanding their structural and/or functional studies. Often, widely accepted mimetic systems have limitations that prevent the study of some membrane proteins. Micelles, bicelles, and liposomes are common biomimetic systems but have problems with membrane compatibility, limited lipid composition, and heterogeneity. To overcome these limitations, polymersomes and hybrid vesicles have become popular alternatives. Polymersomes form from amphiphilic triblock or diblock copolymers and are considered more robust than liposomes. Hybrid vesicles are a combination of lipids and block copolymers that form vesicles composed of a mixture of the two. These hybrid vesicles are appealing because they have the native lipid environment of bilayers but also the stability and customizability of polymersomes. Gramicidin A was incorporated into these polymersomes and characterized using continuous wave electron paramagnetic resonance (CW-EPR) and transmission electron microscopy (TEM). EPR spectroscopy is a powerful biophysical technique used to study the structure and dynamic properties of membrane proteins in their native environment. Spectroscopic studies of gramicidin A have been limited to liposomes; in this study, the membrane peptide is studied in both polymersomes and hybrid vesicles using CW-EPR spectroscopy. Lineshape analysis of spin-labeled gramicidin A revealed linewidth broadening, suggesting that the thicker polymersome membranes restrict the motion of the spin label more when compared to liposome membranes. Statement of Significance: Understanding membrane proteins' structures and functions is critical in the study of many diseases. In order to study them in a native environment, membrane mimetics must be developed that can be suitable for obtaining superior biophysical data quality to characterize structural dynamics while maintaining their native functions and structures. Many currently widely accepted methods have limitations, such as a loss of native structure and function, heterogeneous vesicle formation, restricted lipid types for the vesicle formation for many proteins, and experimental artifacts, which leaves rooms for the development of new biomembrane mimetics. The triblock and diblock polymersomes and hybrid versicles utilized in this study may overcome these limitations and provide the stability and customizability of polymersomes, keeping the biocompatibility and functionality of liposomes for EPR studies of membrane proteins.
在天然环境中研究膜蛋白对于理解其结构和/或功能研究至关重要。通常，广泛接受的模拟系统存在局限性，阻碍了对某些膜蛋白的研究。胶束、双分子层膜囊泡和脂质体是常见的仿生系统，但存在膜兼容性、脂质组成有限和异质性等问题。为了克服这些局限性，聚合物囊泡和混合囊泡已成为受欢迎的替代方案。聚合物囊泡由两亲性三嵌段或二嵌段共聚物形成，被认为比脂质体更坚固。混合囊泡是脂质和嵌段共聚物的组合，形成由两者混合物组成的囊泡。这些混合囊泡很有吸引力，因为它们具有双层膜的天然脂质环境，同时也具有聚合物囊泡的稳定性和可定制性。短杆菌肽A被掺入这些聚合物囊泡中，并使用连续波电子顺磁共振（CW-EPR）和透射电子显微镜（TEM）进行表征。EPR光谱是一种强大的生物物理技术，用于研究膜蛋白在其天然环境中的结构和动态特性。对短杆菌肽A的光谱研究仅限于脂质体；在本研究中，使用CW-EPR光谱在聚合物囊泡和混合囊泡中研究了膜肽。自旋标记的短杆菌肽A的线形分析显示线宽变宽，这表明与脂质体膜相比，较厚的聚合物囊泡膜对自旋标记的运动限制更大。意义声明：了解膜蛋白的结构和功能在许多疾病的研究中至关重要。为了在天然环境中研究它们，必须开发出能够在保持其天然功能和结构的同时，适合获得高质量生物物理数据以表征结构动力学的膜模拟物。目前许多广泛接受的方法存在局限性，例如天然结构和功能的丧失、异质囊泡形成、许多蛋白质形成囊泡时脂质类型受限以及实验假象，这为新型生物膜模拟物的开发留下了空间。本研究中使用的三嵌段和二嵌段聚合物囊泡以及混合囊泡可能克服这些局限性，并提供聚合物囊泡的稳定性和可定制性，同时保持脂质体的生物相容性和功能性，用于膜蛋白的EPR研究。
