Department of Chemistry, Stanford University, Stanford, CA, USA.
Wu-Tsai Neuroscience Institute and ChEM-H institute, Stanford University, Stanford, CA, USA.
Biomater Sci. 2023 Jul 25;11(15):5205-5217. doi: 10.1039/d2bm01856j.
The cell membrane is characterized by a rich variety of topographical features such as local protrusions or invaginations. Curvature-sensing proteins, including the Bin/Amphiphysin/Rvs (BAR) or epsin N-terminal homology (ENTH) family proteins, sense the bending sharpness and the positive/negative sign of these topographical features to induce subsequent intracellular signaling. A number of assays have been developed to study curvature-sensing properties of proteins , but it is still challenging to probe low curvature regime with the diameter of curvature from hundreds of nanometers to micrometers. It is particularly difficult to generate negative membrane curvatures with well-defined curvature values in the low curvature regime. In this work, we develop a nanostructure-based curvature sensing (NanoCurvS) platform that enables quantitative and multiplex analysis of curvature-sensitive proteins in the low curvature regime, in both negative and positive directions. We use NanoCurvS to quantitatively measure the sensing range of a negative curvature-sensing protein IRSp53 (an I-BAR protein) and a positive curvature-sensing protein FBP17 (an F-BAR protein). We find that, in cell lysates, the I-BAR domain of IRSp53 is able to sense shallow negative curvatures with the diameter-of-curvature up to 1500 nm, a range much wider than previously expected. NanoCurvS is also used to probe the autoinhibition effect of IRSp53 and the phosphorylation effect of FBP17. Therefore, the NanoCurvS platform provides a robust, multiplex, and easy-to-use tool for quantitative analysis of both positive and negative curvature-sensing proteins.
细胞膜的特点是具有丰富的地形特征，如局部突起或内陷。曲率感应蛋白，包括 Bin/Amphiphysin/Rvs（BAR）或 epsin N 端同源（ENTH）家族蛋白，感知这些地形特征的弯曲锐度和正负符号，以诱导后续的细胞内信号转导。已经开发了许多测定法来研究蛋白质的曲率感应特性，但是仍然难以探测直径从数百纳米到数微米的低曲率范围。在低曲率范围内，很难生成具有明确定义曲率值的负膜曲率。在这项工作中，我们开发了一种基于纳米结构的曲率感应（NanoCurvS）平台，该平台能够在低曲率范围内定量分析正负曲率敏感蛋白。我们使用 NanoCurvS 定量测量了负曲率感应蛋白 IRSp53（I-BAR 蛋白）和正曲率感应蛋白 FBP17（F-BAR 蛋白）的感应范围。我们发现，在细胞裂解物中，IRSp53 的 I-BAR 结构域能够感应浅的负曲率，曲率直径可达 1500nm，范围比之前预期的要宽得多。NanoCurvS 还用于探测 IRSp53 的自动抑制效应和 FBP17 的磷酸化效应。因此，NanoCurvS 平台为定量分析正负曲率感应蛋白提供了一种强大、多功能且易于使用的工具。
