Multivalent Ligand-Receptor Binding on Lipid Bilayers

描述(申请人提供)：本提案中研究的长期重点是了解脂膜成分在多价配体/受体结合中的作用。模型双层系统的最新发展表明，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。关于这些“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，它们可能会显著增强多价配体-受体的附着。其机制可能包括将膜结合的配体集中到小的高度集中的区域，或者改变配体的方向以使与外部蛋白质的结合更有利。膜的组成和取向可以直接影响多价结合的平衡解离常数的假设将被用来检验免疫球蛋白抗体(二价系统)和霍乱毒素(五价系统)。将使用新开发的高通量微流控策略来探索初始结合和随后的侧向结合。这些基础研究最终可能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。这项建议的四个具体目的包括：(1)研究抗2，4-DNP免疫球蛋白与其脂联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，因为这可能会迫使配体离开膜；因此，使其更容易获得。(2)对抗体的第二离解常数进行研究。这将作为胆固醇含量、配体密度和脂尾中不饱和部位的函数来完成。一个中心假设是，减弱膜扩散常数的成分也可能阻碍侧向结合作用。(3)研究脂筏形成对结合的影响。胆固醇/鞘磷脂/磷脂的混合物将被探索它们影响膜中初始和后续结合作用的能力。如果可能，将在富含磷脂和富鞘磷脂的两个相中进行结合测量。(4)上述三个目标将扩大至霍乱毒素/GM1。这个系统应该表现出截然不同的行为，因为已知GM1形成簇，而不是作为膜的理想稀释组分。在临界浓度以上，结合常数可能会发生显著变化。

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.