Multivalent Ligand-Receptor Binding on Lipid Bilayers

脂质双层上的多价配体-受体结合

• 批准号: 6872900
• 负责人: Paul Cremer
• 金额: $ 25.06万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6872900
• 关键词: 2,4 dinitrophenol; binding sites; cholera toxin; cholesterol; fluid flow; haptens; high throughput technology; immunoglobulin G; ligands; lipid bilayer membrane; membrane lipids; phospholipids; receptor binding; sphingolipids

DESCRIPTION (provided by applicant): The long-term focus of the research in this proposal is to understand the role of lipid membrane composition on multivalent ligand/receptor binding. Recent developments with model bilayer systems have shown that structured liquid-ordered regions rich in cholesterol and sphingolipids can coexist along side a phospholipid rich, liquid-disordered domain. It is still controversial as to whether these "lipid rafts" also exist in cells; however, there has been speculation that if they do exist in vivo, they could significantly enhance multivalent ligand-receptor attachment. The mechanism might involve either concentrating membrane bound ligands into small highly concentrated regions or changing the ligand orientation to make binding with external proteins more favorable. The hypothesis that membrane composition and orientation can directly affect the equilibrium dissociation constants of multivalent binding will be tested for IgG antibodies (a bivalent system) and for cholera toxin (a pentavalent system). Both initial binding as well as subsequent lateral binding will be explored using newly developed high-throughput microfluidic strategies. Such fundamental studies may ultimately play a role in developing inhibitors to the initial step of pathogen entry into cells. The four specific aims of this proposal include: (1) investigating the initial binding event for anti-2,4 DNP IgG with its lipid conjugated hapten. Studies will be conducted as a function of cholesterol content as this may force the ligand out of the membrane; hence, making it more available. (2) Investigations of the second dissociation constant for antibodies will be conducted. This will be done as a function of cholesterol content, ligand density, and sites of unsaturation in the lipid tails. One central hypothesis is that components which attenuate the diffusion constant of the membrane may also impede lateral binding interactions. (3) The effect of lipid raft formation on binding will be undertaken. Mixtures of cholesterol/sphingolipid/phospholipid will be probed for their ability to affect both the initial and subsequent binding interactions in membranes. Measurements of binding will be made in both the phospholipid-rich and sphingolipid-rich phases when possible. (4) The above three aims will be expanded to cholera toxin/GM1. This system should show dramatically different behavior because GM1 is known to form clusters, rather than acting as an ideal dilute constituent of the membrane. The binding constant may change dramatically above a critical concentration.

描述（由申请人提供）：本提案研究的长期重点是了解脂质膜成分对多价配体/受体结合的作用。模型双层系统的最新进展表明，富含胆固醇和鞘脂的结构化液体有序区域可以与富含磷脂的液体无序区域共存。这些“脂筏”是否也存在于细胞中仍存在争议。然而，有人推测，如果它们确实存在于体内，它们可以显着增强多价配体-受体的附着。该机制可能涉及将膜结合配体浓缩到小的高度浓缩区域或改变配体方向以使与外部蛋白质的结合更有利。膜组成和方向可以直接影响多价结合的平衡解离常数的假设将针对 IgG 抗体（二价系统）和霍乱毒素（五价系统）进行测试。将使用新开发的高通量微流体策略来探索初始结合以及随后的横向结合。这些基础研究可能最终在开发病原体进入细胞的第一步抑制剂方面发挥作用。该提案的四个具体目标包括：(1) 研究抗 2,4 DNP IgG 与其脂质缀合半抗原的初始结合事件。研究将根据胆固醇含量进行，因为这可能迫使配体脱离膜；因此，使其更可用。 (2) 对抗体的第二解离常数进行研究。这将根据胆固醇含量、配体密度和脂质尾部不饱和位点来完成。一个中心假设是，减弱膜扩散常数的成分也可能阻碍横向结合相互作用。 (3)将研究脂筏形成对结合的影响。将探测胆固醇/鞘脂/磷脂的混合物影响膜中初始和随后的结合相互作用的能力。如果可能，将在富含磷脂和富含鞘脂的相中进行结合测量。 (4) 上述三个目标将扩展到霍乱毒素/GM1。该系统应该表现出显着不同的行为，因为已知 GM1 会形成簇，而不是充当膜的理想稀释成分。超过临界浓度时，结合常数可能会发生显着变化。