Protein-mediated molecular adhesion: AFM studies

蛋白质介导的分子粘附：AFM 研究

• 批准号: 6788712
• 负责人: VINCENT T MOY
• 金额: $ 24.98万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6788712
• 关键词: T lymphocyte; antigen presenting cell; atomic force microscopy; cell adhesion; cell adhesion molecules; conformation; fluorescence resonance energy transfer; integrins; leukocyte activation /transformation; leukocyte adhesion molecules; molecular dynamics; protein protein interaction; protein structure; receptor binding

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.

描述（由申请人提供）：白细胞整合素、淋巴细胞功能相关抗原 1 (LFA-1) 与其天然配体、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 细胞激活过程中稳定 T 淋巴细胞和抗原呈递细胞的相互作用。在此过程中被认为重要的 LFA-1/ICAM-1 相互作用的生物物理特性包括复合物在高亲和力状态和低亲和力状态之间调节的能力以及 LFA-1/ICAM-1 复合物的内在机械强度。直到最近，粘附仍通过涉及动力学测量或简单细胞粘附测定的间接方法进行检查。现在，原子力显微镜（AFM）的进步已经能够在单个配体-受体对水平上直接测量粘附力。 AFM 测量与诱变实验相结合，可用于识别导致 LFA-1/ICAM-1 复合物解离电位主要特征的分子决定因素。 在这里，我们建议获得 AFM 力测量，以研究在初始 T 细胞/APC 接触期间以及在粘附进一步加强时的后续 T 细胞激活期间 LFA-1 和 ICAM-1 之间相互作用的机制。前三个目标将测量动态强度并确定 LFA-1/ICAM-1 相互作用的结构成分，最后两个目标将探索 T 细胞激活后增强结合的机制。这些实验的结果将回答以下问题：1) LFA-1/ICAM-1 复合物如何解绑？ 2) LFA-1/ICAM-1复合物的结合强度如何随LFA-1的构象变化？ 3) 允许LFA-1/ICAM-1复合物抵抗大拉力的分子决定因素是什么？ 4) 细胞活化后淋巴细胞粘附增强是否是由于受体聚集所致？ 5) 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。