Protein-mediated molecular adhesion: AFM studies

描述（由申请方提供）：白细胞整联蛋白、淋巴细胞功能相关抗原-1（LFA-1）及其天然配体、细胞间粘附分子-1（ICAM-1）之间的粘附相互作用对于免疫系统的正常功能至关重要。它在T细胞活化过程中稳定T淋巴细胞和抗原呈递细胞的相互作用。LFA-1/ICAM-1相互作用的生物物理性质在该过程中被认为是重要的，包括复合物在高和低亲和力状态之间调节的能力和LFA-1/ICAM-1复合物的固有机械强度。直到最近，粘附通过间接的方法，涉及动力学测量或简单的细胞粘附测定。现在，原子力显微镜（AFM）的进步已经能够在单个配体-受体对的水平上直接测量粘附力。AFM测量，结合诱变实验，可用于确定的分子决定因素，负责的主要功能的解离电位的LFA-1/ICAM-1复合物。 在这里，我们建议获得AFM力测量，以调查的机制，有助于LFA-1和ICAM-1之间的相互作用，在初始的T细胞/APC接触，并在随后的T细胞活化时，粘附进一步加强。前三个目标将测量动态强度并确定LFA-1/ICAM-1相互作用的结构组分，最后两个目标将探索T细胞活化后增强结合的机制。这些实验的结果将回答以下问题：1）LFA-1/ICAM-1复合物如何解结合？2)LFA-1/ICAM-1复合物的结合强度如何随LFA-1的构象而变化？3)是什么样的分子决定因素使LFA-1/ICAM-1复合物能够抵抗大的拉力？4)细胞活化后淋巴细胞粘附增强是由于受体聚集吗？ICAM-1的二聚体结构是否增强了其与LFA-1的相互作用？最终，这些实验将帮助我们更好地理解决定配体-受体结合强度的生物物理机制，并有助于开发免疫系统相关疾病的治疗方法。

DESCRIPTION (provided by applicant): The adhesive interactions between the leukocyte integrin, lymphocyte function associated antigen-1 (LFA-1) and its native ligand, intercellular adhesion molecule-1 (ICAM-1) are crucial for normal function of the immune system. It stabilizes the interactions of T lymphocytes and antigen-presenting cells during the process of T cell activation. The biophysical properties of the LFA-1/ICAM-1 interaction that are deemed important in this process include the ability of the complex to modulate between high and low affinity states and the intrinsic mechanical strength of the LFA-1/ICAM-1 complex. Until recently adhesion was examined through indirect methods that involved kinetic measurements or simple cell adhesion assays. Now, advances in atomic force microscopy (AFM) have enabled direct measurements of adhesive forces at the level of single ligand-receptor pairs. The AFM measurements, when combined with mutagensis experiments, can be used to identify the molecular determinants that are responsible for the major features in the dissociation potential of the LFA-1/ICAM-1 complex. Here, we propose to acquire AFM force measurements to investigate the mechanisms that contribute to the interaction between LFA-1 and ICAM-1 during initial T-celI/APC contact and during subsequent T-cell activation when adhesion is further strengthened. The first three objectives will measure the dynamic strength and identify the structural components of the LFA-1/ICAM-1 interaction and the last two objectives will explore mechanisms for the enhanced binding following T-cell activation. Results from these experiments will answer the following questions: 1) How does the LFA-1/ICAM-1 complex unbind? 2) How does the bond strength of the LFA-1/ICAM-1 complex change with the conformation of LFA-1? 3) What are the molecular determinants that permit the LFA-1/ICAM-1 complex to resist large pulling forces? 4) Is enhanced lymphocyte adhesion following cell activation due to receptor clustering? and 5) Does the dimeric structure of ICAM-1 strengthen its interaction with LFA-1? Ultimately, these experiments will help us achieve a better understanding of the biophysical mechanisms that determine ligand-receptor binding strength and could aid in the development of treatments for immune system related disorders.