Multiplexed Platform to Probe Interactions at the Model Cell Membrane Interface

描述(申请人提供)：可溶性蛋白是许多生化信号通路的关键媒介，通过直接与脂质双层相互作用和通过膜结合的受体。细胞膜的成分参与许多重要的生物学过程，包括凝血、病毒感染和信号转导，因此，它们是治疗药物的常见靶点。因此，发展分析方法来研究细胞膜的相互作用是至关重要的。这一提议集成了两项关键技术，硅光子微环谐振器阵列和磷脂双层纳米盘，它们一起允许多路筛选可溶蛋白质与脂类和膜包埋靶标的相互作用。微环谐振器阵列是一种本质上可多路复用的、无标记的分析平台，以前已被应用于研究蛋白质-蛋白质、蛋白质-核酸和核酸-核酸相互作用。纳米盘是蛋白质稳定的脂质组装体，代表了一种方便的结构来模拟天然的磷脂双层，研究膜组成的影响，并溶解膜包埋的靶标。融合在一起，这些技术将提供无与伦比的能力，以高通量、多路和信息丰富的分析形式询问膜-配体相互作用的生物物理形式。这项拟议工作的生物学动机是凝血，这是一个关键的调节过程，是血栓形成和出血性疾病治疗干预的目标。除了蛋白质-膜蛋白相互作用外，凝血因子在细胞膜表面的组装还受到蛋白质-脂相互作用的调节，特别是与严重依赖于二价金属离子的阴离子脂类的相互作用。然而，血液凝结中蛋白质-脂质相互作用的许多细节仍然知之甚少，而对基础知识的改进可能会揭示威胁生命的血栓性疾病的新治疗策略。在这里，我们建议通过使用与纳米盘功能化的微环谐振器的多路阵列来阐明凝血因子(分别为因子VIIa和X；FVIIa和Fx)与凝血抑制物(激活蛋白C；APC)的复杂的脂类和阳离子依赖的结合，这些微环谐振器呈现可变但定义明确的脂类成分。我们还将实现一种新的溶液相梯度标记器，以动态控制溶液中金属离子的浓度和特性，同时实时监测这些变化对蛋白质-脂质相互作用的影响。总而言之，这些研究将提供前所未有的途径，在模型界面上高通量和多变量地询问血液凝固过程，更广泛地说，将验证纳米盘和微环谐振器阵列的集成，以用于细胞膜相互作用的高度多路研究。

DESCRIPTION (provided by applicant): Soluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes including blood coagulation, viral infection, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. This proposal integrates two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer Nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Fused together, these technologies will offer an unsurpassed capacity to interrogate the biophysical modalities of membrane-ligand interactions in high-throughput, multiplexed, and information rich assay formats. The biological motivation for the proposed work is blood coagulation, which is a critical regulatory process that is a target for therapeutic intervention i thrombosis and bleeding disorders. In addition to protein- membrane protein interactions, the assembly of coagulation factors at the cell membrane surface is additionally regulated by protein-lipid interactions, and in particular interactions with anionic lipids that are critically-dependent upon divalent metal ions. However many details of protein-lipid interactions in blood clotting remain poorly understood, and an improved fundamental understanding might reveal new therapeutic strategies for life-threatening thrombotic diseases. Herein we propose to shed light onto complex lipid- and cation-dependent binding of blood coagulation factors (Factors VIIa and X; fVIIa and fX, respectively) and clotting inhibitors (activated protein C; aPC) through the use of multiplexed arrays of microring resonators functionalized with Nanodiscs that present variable, yet well-defined lipid composition. We also will implement a novel solution-phase gradient maker to dynamically control the concentration and identity of metal cations in solution while simultaneously monitoring the effects of these changes on protein-lipid interactions in real time. Together, these studies will provide unprecedented access to high throughput and multivariate interrogation of blood coagulation processes at model interfaces, and more generally will validate the integration of Nanodiscs and microring resonator arrays for highly multiplexed studies of interactions at the cell membrane.