Walters Sara H, Signorelli Rachel L, Payne Allyson G, Hojjatian Alimohammad, Fuglestad Brian
Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia, USA.
Office of the Vice President for Research and Innovation, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Soft Matter. 2025 May 7;21(18):3547-3557. doi: 10.1039/d5sm00033e.
The study of membranes and their associated proteins is critical for understanding cellular processes. investigations utilizing membrane models often have limitations in their biological relevance due to the dissimilarity of experimentally compatible membrane mimetics to biological membranes. Development of membrane models that better mimic cellular membranes enables more biologically accurate observations of membrane associated proteins. In this work, we present upper tolerance concentrations for a range of lipids incorporated into reverse micelles (RMs), confirmed with dynamic light scattering (DLS). A breadth of lipid incorporation enabled biologically inspired RMs to be formulated based on the molar ratios of lipids present in eukaryotic membrane leaflets. Three systems were formulated matching lipid compositions of the inner leaflet of the plasma membrane (PM-RM), the outer mitochondrial membrane leaflet (MI-RM), and the outer rough endoplasmic reticulum membrane leaflet (ER-RM). The biologically-inspired RM formulations were characterized using DLS and cryo-electron microscopy (cryo-EM) and were found to have favorable properties for protein encapsulation. All three biologically inspired RM formulations effectively encapsulated fatty acid binding protein 4 (FABP4), a protein which shuttles fatty acids between membranes, confirmed by NMR. Also presented in this work is the first known high-resolution observation of the membrane-bound state of sterol carrier protein 2 (SCP2), a protein responsible for transporting an array of lipids between membranes. SCP2 was successfully encapsulated within all three RM systems, enabling NMR observation of the membrane interface of SCP2. The tolerances and formulations reported here allow for tailoring of RMs to mimic specific cellular membranes and will enhance studies of protein interactions with lipids and membranes among other investigations.
对膜及其相关蛋白质的研究对于理解细胞过程至关重要。由于实验上兼容的膜模拟物与生物膜存在差异，利用膜模型的研究在生物学相关性方面往往存在局限性。开发能更好模拟细胞膜的膜模型，可实现对膜相关蛋白质更具生物学准确性的观察。在这项工作中，我们给出了一系列掺入反胶束（RMs）中的脂质的耐受浓度上限，通过动态光散射（DLS）得以证实。广泛的脂质掺入使得能够基于真核细胞膜小叶中脂质的摩尔比来配制受生物学启发的RMs。配制了三个系统，分别匹配质膜内小叶（PM-RM）、线粒体外膜小叶（MI-RM）和糙面内质网外膜小叶（ER-RM）的脂质组成。使用DLS和冷冻电子显微镜（cryo-EM）对受生物学启发的RMs配方进行了表征，发现它们具有有利于蛋白质包封的特性。通过核磁共振（NMR）证实，所有三种受生物学启发的RMs配方都有效地包封了脂肪酸结合蛋白4（FABP4），该蛋白在膜之间穿梭运输脂肪酸。这项工作还首次给出了对甾醇载体蛋白2（SCP2）膜结合状态的高分辨率观察，SCP2是一种负责在膜之间运输一系列脂质的蛋白质。SCP2成功地封装在所有三个RM系统中，使得能够通过NMR观察SCP2的膜界面。这里报道的耐受性和配方允许定制RMs以模拟特定的细胞膜，并将加强对蛋白质与脂质及膜之间相互作用的研究以及其他研究。
