Protein-mediated molecular adhesion: AFM studies

蛋白质介导的分子粘附：AFM 研究

• 批准号: 6680701
• 负责人: VINCENT T MOY
• 金额: $ 18.88万
• 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/6680701
• 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）之间的粘合性相互作用对于免疫系统的正常功能至关重要。它稳定在T细胞激活过程中T淋巴细胞和抗原呈递细胞的相互作用。在此过程中认为重要的LFA-1/ICAM-1相互作用的生物物理特性包括复合物调节高亲和力和低亲和力之间的能力以及LFA-1/ICAM-1复合物的固有机械强度。直到最近，通过间接方法检查了粘附，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）的进步已可以直接测量单个配体受体对水平的粘合力。当与诱变实验结合使用时，AFM测量值可用于识别负责LFA-1/ICAM-1复合物离解势的主要特征的分子决定簇。 在这里，我们建议获取AFM力测量值，以研究在初始T-Celi/APC接触期间以及随后的T细胞激活期间，在进一步加强粘附时随后的T细胞激活期间LFA-1和ICAM-1之间的相互作用的机制。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复合物如何解开？ 2）LFA-1/ICAM-1复合物的键强度如何随LFA-1的构象变化？ 3）什么是允许LFA-1/ICAM-1复合物抵抗大拉力的分子决定因素？ 4）由于受体聚类引起的细胞激活后，淋巴细胞粘附是否会增强？ 5）ICAM-1的二聚体结构是否可以增强其与LFA-1的相互作用？最终，这些实验将有助于我们更好地了解确定配体 - 受体结合强度的生物物理机制，并可以帮助开发免疫系统相关疾病的治疗方法。