Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence
Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence
复制标题
DOI：
10.1016/j.bpj.2019.12.002
复制
发表时间：
2020-01-21
影响因子：
3.4
通讯作者：
Mueller, Joachim D.
中科院分区：
文献类型：
--
作者：
Angert, Isaac;Karuka, Siddarth Reddy;Mueller, Joachim D.
质膜（PM）上微妙和短暂的蛋白质接合对于广泛的细胞功能至关重要，包括细胞运动，信号转导和病毒复制。在这里，我们描述了一个双色（DC）的荧光Z-扫描技术，这已被证明是成功的外周膜蛋白结合到活细胞中的PM的定量扩展。我们证明，共表达的第二个，明显着色的荧光蛋白提供了一个可溶性的参考物种，描绘的程度的细胞质和降低检测阈值的z-扫描PM结合测量的数量级。DC z扫描为每个检测通道生成强度分布，其包含关于外周膜和参考蛋白的轴向分布的信息。DC z扫描的拟合模型的开发和验证使用简单的模型系统。接下来，我们应用定量DC Z-扫描技术来研究两种外周膜蛋白系统的结合，其中先前的Z-扫描研究未能检测到结合：人类免疫缺陷病毒1型（HIV-1）基质（MA）蛋白和成纤维细胞生长因子受体底物2 α的脂质缺陷突变体。我们的研究结果表明，这些突变严重破坏PM协会的成纤维细胞生长因子受体底物2 α，但不消除it. We进一步检测HIV-1 MA结合PM使用DC z扫描。有趣的是，我们的数据表明，HIV-1 MA与PM协同结合，解离系数K-d类似于16 μ M，希尔系数n类似于2。
Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2 alpha. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2 alpha but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of K-d similar to 16 mu M and Hill coefficient of n similar to 2.