Multivalent Ligand-Receptor Binding on Lipid Bilayers

描述（由申请人提供）：本课题的长期研究重点是了解脂质膜组成在多价配体/受体结合中的作用。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。至于这些“脂筏”是否也存在于细胞中，目前仍有争议；然而，有人猜测，如果它们确实存在于体内，它们可以显著增强多价配体-受体的附着。其机制可能包括将膜结合的配体浓缩成小的高浓度区域或改变配体的取向以使其更有利于与外部蛋白质的结合。假设膜的组成和取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）。初始结合和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抑制病原体进入细胞的初始步骤中发挥作用。本提案的四个具体目标包括：(1)研究抗2,4 DNP IgG与其脂质偶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脱离膜；因此，使它更容易获得。(2)对抗体的第二解离常数进行研究。这将作为胆固醇含量、配体密度和脂质尾部不饱和位点的函数来完成。一个中心假设是，减弱膜扩散常数的成分也可能阻碍横向结合相互作用。(3)研究脂筏形成对结合的影响。将探讨胆固醇/鞘脂/磷脂的混合物对膜内初始和后续结合相互作用的影响。如果可能的话，结合的测量将在富含磷脂和富含鞘脂的阶段进行。(4)上述三个目标将扩大到霍乱毒素/GM1。这个系统应该表现出显著不同的行为，因为GM1是已知的形成团簇，而不是作为膜的理想稀释成分。在临界浓度以上，结合常数可能发生显著变化。

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.