Malinick Alexander S, Stuart Daniel D, Ebel Cole P, Van Zant Westley, Rizvi David, Gutierrez Jasmine, Cheng Quan
Department of Chemistry, University of California, Riverside, CA, 92521, USA.
Department of Chemistry, University of California, Riverside, CA, 92521, USA.
Biosens Bioelectron. 2025 Nov 1;287:117713. doi: 10.1016/j.bios.2025.117713. Epub 2025 Jun 18.
Curvature-sensitive proteins play important roles in cellular physiology. To study their interaction properties, reliable platforms of curved membrane mimics are key but current systems are limited by the requirement for fluorescent labels, which can modify membrane conformation and activity, leading to obscured description of interaction behavior as compared to native conditions. We report here the construction and application of a robust and tunable curved membrane interface for label-free surface plasmon resonance (SPR) based probing of the interactions with bridging integrator 1 (BIN1), a BAR-domain containing protein that has been linked to cancer and autoimmune diseases. The curvature was tuned with varying vesicle size, and surface charge was controlled by doping different content of ganglioside GM1. A twentyfold enhancement of BIN1 response was observed on 50-nm vesicles as compared to the flat SLB. Replacement of GM with neutral GA or reducing GM content from 1 % to 0.1 % in vesicles led to much lower response signal, suggesting the preference of negatively charged membranes in BIN1 binding. To demonstrate the application in complex media and for biomarker monitoring, we measured BIN1 in spiked urine samples, which showed excellent results. Small loss in signal was observed as compared to that in PBS buffer, which have been attributed to ionic compounds present in the urine sample that interfere with BIN1-membrane interactions. This work shows that the vesicle-based curved membrane proves to be an effective platform for investigating membrane-curvature sensing proteins and their biophysical interactions, and for quantifying protein markers in biological matrices by SPR.
曲率敏感蛋白在细胞生理学中发挥着重要作用。为了研究它们的相互作用特性，可靠的弯曲膜模拟平台是关键，但目前的系统受到荧光标记需求的限制，荧光标记会改变膜的构象和活性，导致与天然条件相比，相互作用行为的描述变得模糊。我们在此报告了一种强大且可调谐的弯曲膜界面的构建和应用，该界面用于基于无标记表面等离子体共振（SPR）探测与桥连整合蛋白1（BIN1）的相互作用，BIN1是一种含有BAR结构域的蛋白质，与癌症和自身免疫性疾病有关。通过改变囊泡大小来调节曲率，并通过掺杂不同含量的神经节苷脂GM1来控制表面电荷。与平坦的支持脂质双分子层（SLB）相比，在50纳米的囊泡上观察到BIN1的响应增强了20倍。用中性的神经节苷脂GA替代GM或使囊泡中的GM含量从1%降至0.1%会导致响应信号大幅降低，这表明BIN1结合时偏好带负电荷的膜。为了证明在复杂介质中的应用以及用于生物标志物监测，我们测量了加标的尿液样本中的BIN1，结果显示良好。与在磷酸盐缓冲盐水（PBS）缓冲液中的情况相比，观察到信号有小幅损失，这归因于尿液样本中存在的离子化合物干扰了BIN1与膜的相互作用。这项工作表明，基于囊泡的弯曲膜被证明是研究膜曲率传感蛋白及其生物物理相互作用以及通过SPR定量生物基质中蛋白质标志物的有效平台。
