Characterization of Gramicidin A in Triblock and Diblock Polymersomes and Hybrid Vesicles via Continuous Wave Electron Paramagnetic Resonance Spectroscopy.

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.