Multivalent Ligand-Receptor Binding on Lipid Bilayers

脂质双层上的多价配体-受体结合

• 批准号: 7228928
• 负责人: Paul Cremer
• 金额: $ 22.81万
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228928
• 关键词: 2,4-Dinitrophenol; Affect; Antibodies; Attenuated; Behavior; Binding; Binding Sites; Biological Models; Cell surface; Cells; Ceramides; Chemistry; Cholera Toxin; Cholera Toxin Protomer B; Cholesterol; DNA Sequence Rearrangement; Data; Defect; Development; Diffusion; Disease; Dissociation; Equilibrium; Event; Extracellular Protein; Fab Immunoglobulins; Ganglioside GM1; Haptens; Immunoglobulin G; Individual; Investigation; Laboratories; Lateral; Lead; Lecithin; Ligand Binding; Ligands; Link; Lipid Bilayers; Lipids; Liquid substance; Measurement; Membrane; Membrane Lipids; Membrane Microdomains; Microfluidics; Microscopy; Modeling; Phase; Phospholipids; Play; Proteins; Research; Role; Side; Site; Spectrum Analysis; Sphingolipids; Structure; Surface; System; Tail; Testing; density; dinitrophenyl; in vivo; inhibitor/antagonist; interest; pathogen; receptor; receptor binding; research study; two-dimensional

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）：本提案中研究的长期重点是了解脂质膜组成对多价配体/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沿着富含磷脂的液体无序结构域共存。对于这些“脂筏”是否也存在于细胞中仍然存在争议;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-受体附着。该机制可能涉及将膜结合的配体浓缩到小的高度浓缩的区域中或改变配体取向以使与外部蛋白质的结合更有利。假设膜的组成和方向可以直接影响多价结合的平衡解离常数将测试IgG抗体（二价系统）和霍乱毒素（五价系统）。初始结合以及随后的横向结合将使用新开发的高通量微流体策略进行探索。这些基础研究可能最终在开发病原体进入细胞的初始步骤的抑制剂方面发挥作用。本研究的四个具体目标包括：（1）研究抗2，4 DNP IgG与其脂质结合半抗原的初始结合事件。研究将作为胆固醇含量的函数进行，因为这可能迫使配体离开膜;因此，使其更可用。(2)将对抗体的第二解离常数进行研究。这将作为胆固醇含量、配体密度和脂质尾中不饱和位点的函数来完成。一个中心假设是，衰减膜的扩散常数的组分也可能阻碍横向结合相互作用。(3)脂筏形成对结合的影响将进行。将探测胆固醇/鞘脂/磷脂的混合物影响膜中初始和随后结合相互作用的能力。如可能，将在富磷脂和富鞘脂相中进行结合测量。(4)上述三个目标将扩大到霍乱毒素/GM 1。这个系统应该表现出显着不同的行为，因为GM 1是已知的形成集群，而不是作为一个理想的膜的稀释成分。结合常数可能会在临界浓度以上发生显著变化。