Multiplexed Platform to Probe Interactions at the Model Cell Membrane Interface

用于探测模型细胞膜界面相互作用的多重平台

• 批准号: 8674700
• 负责人: Ryan C Bailey
• 金额: $ 29.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8674700
• 关键词: Affinity; Antibodies; Binding; Biochemical; Biological; Biological Assay; Biological Process; Blood Coagulation Factor; Blood coagulation; Cations; Cell membrane; Coagulation Process; Complex; DNA; Dependence; Development; Disease; Electrostatics; Ensure; Equilibrium; Ethanolamines; Factor VIIa; Hemorrhage; Ions; Kinetics; Label; Lecithin; Life; Ligands; Light; Lipid Bilayers; Lipids; Mediating; Mediation; Mediator of activation protein; Membrane; Membrane Lipids; Membrane Proteins; Metals; Methods; Modality; Modeling; Monitor; Motivation; Nucleic Acids; Oxides; Phase; Phosphatidic Acid; Phosphatidylserines; Phospholipids; Polymers; Process; Protein C Inhibitor; Proteins; Prothrombin; Sampling; Signal Pathway; Signal Transduction; Silicon; Solutions; Surface; Technology; Therapeutic Agents; Therapeutic Intervention; Thrombosis; Time; Virus Diseases; Work; base; blood coagulation process; divalent metal; improved; metal complex; nanodisk; novel; novel therapeutics; photonics; public health relevance; receptor binding; response; screening; self assembly; sensor

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.

描述（由申请人提供）：可溶性蛋白质是许多生化信号传导途径的关键介质，通过与脂质双层直接相互作用和通过膜结合受体。细胞膜的组分参与许多重要的生物过程，包括血液凝固、病毒感染和信号转导，因此，它们是治疗剂的常见靶标。因此，开发分析方法来研究细胞膜上的相互作用至关重要。该提案集成了两项关键技术，即硅光子微谐振器阵列和磷脂双层纳米盘，它们共同允许对可溶性蛋白质与脂质和膜嵌入靶的相互作用进行多重筛选。Microbial共振器阵列是一种本质上可复用的、无标记的分析平台，其先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，其代表了模拟天然磷脂双层、研究膜组合物的影响以及溶解膜包埋的靶标的便利构建体。融合在一起，这些技术将提供一个无与伦比的能力，询问生物物理模式的膜配体相互作用的高通量，多路复用和信息丰富的测定格式。 所提出的工作的生物学动机是血液凝固，这是一个关键的调节过程，是血栓形成和出血性疾病的治疗干预的目标。除了蛋白质-膜蛋白相互作用之外，凝血因子在细胞膜表面的组装还受到蛋白质-脂质相互作用的调节，特别是与关键依赖于二价金属离子的阴离子脂质的相互作用。然而，凝血过程中蛋白质-脂质相互作用的许多细节仍然知之甚少，而基础知识的改进可能会揭示危及生命的血栓性疾病的新治疗策略。 在此，我们提出了阐明复杂的脂质和阳离子依赖性结合的血液凝固因子（因子VIIa和X; fVIIa和fX，分别）和凝血抑制剂（活化蛋白C; aPC）通过使用多路复用阵列的microbial谐振器功能化与纳米盘，目前可变的，但定义明确的脂质组成。我们还将实施一种新的溶液相梯度制造商，以动态控制溶液中金属阳离子的浓度和身份，同时监测这些变化对蛋白质-脂质相互作用的影响，在真实的时间。总之，这些研究将提供前所未有的访问高通量和多变量的血液凝固过程中的模型接口的询问，更一般地将验证纳米盘和microbial谐振器阵列的集成，在细胞膜的相互作用的高度多路复用的研究。