MEMBRANE DYNAMICS AND FUNCTION OF MHC CLASS II MUTANTS

主要组织相容性复合物（MHC）的II类分子是 在T细胞依赖性免疫应答中对抗原呈递至关重要。的 侧位和旋转动力学以及II类的膜分布 这些分子与信号转导和抗原 演示文稿. II类分子α-和β-的细胞质结构域 β链对于这些分子的正常功能至关重要， 膜运动的主要决定因素。 与达特茅斯医学院合作，我们将研究 II类分子内的突变对其膜动力学的影响 和生物功能。表达突变型II类分子的B细胞， 截短和取代的胞质结构域，以及II类 在II类接触残基中具有突变的分子（α- β 2）二聚体，将使用生物物理方法进行检查，以确定是否 特定的II类分子结构特征和结构域在 调节分子动力学和分子间缔合。这些 在调节II类功能中涉及的现象包括 B细胞中的信号转导和抗原呈递给T细胞。 具体目标包括：1）时间分辨磷光各向异性， 偏振荧光衰减和荧光光漂白恢复 研究II类细胞质突变体，以确定 残基调节II类分子的旋转和侧向运动。一个 该目的的另一方面是检查II类分子， 影响信号传导的突变。突变对分子生物学的影响 运动将与信号缺陷相关，并与 蛋白质相互作用的最近邻分析， 和控制II类的膜运动和生理; 2）横向和 旋转扩散研究野生型和突变型II类分子在 是否存在免疫原性肽。这些研究将 检查肽调节细胞中II类聚集的能力， 是否存在II类细胞质结构域 3）检测突变对接触分子的影响， II类（α-β）2中α-β分子之间的氨基酸 二聚体。II类分子聚集似乎需要有效的 信号转导和抗原呈递。这方面的研究将 检查二聚体形成能力的变化，评价为旋转 和能量转移变化的测量，与疗效相关 抗原呈递

Class II molecules of the Major Histocompatibility Complex (MHC) are central to antigen presentation in T cell-dependent immune responses. The lateral and rotational dynamics and membrane distribution of Class II molecules have been implicated in signal transduction and antigen presentation. The cytoplasmic domains of Class II molecule alpha- and beta-chains are critical for normal functioning of these molecules and are major determinants of their membrane motions. In collaboration with Dartmouth Medical School, we will examine the effects of mutations within Class II molecules on their membrane dynamics and biological function. B cells expressing mutant Class II molecules with truncated and substituted cytoplasmic domains, as well as Class II molecules with mutations in the contact residues of the Class II (alpha- beta2) dimer, will be examined using biophysical methods to determine if specific Class II molecular structural features and domains have roles in modulating molecular dynamics and intermolecular associations. These phenomena have been implicated in regulating Class II functions including signal transduction in the B cell and antigen presentation to T cells. Specific aims include: l) Time-resolved phosphorescence anisotropy, polarized fluorescence depletion and fluorescence photobleaching recovery studies on cytoplasmic mutants of Class II to determine which amino acid residues regulate Class II molecular rotational and lateral motions. An additional aspect of this aim is to examine Class II molecules with mutations affecting signalling. The effect of mutations on molecular motions will be correlated with signalling defects and correlated with nearest neighbor analysis of protein-protein interactions which contribute and control Class II's membrane motions and physiology; 2) Lateral and rotational diffusion studies of wild type and mutant Class II molecules in the presence or absence of immunogenic peptides. These studies will examine the ability of peptides to regulate Class II aggregation in the presence or absence of cytoplasmic domain structures of Class II molecules; and 3) Examination of the effect of mutations in the contact amino acids between alpha-beta molecules in the Class II (alpha-beta)2 dimer. Class II molecular aggregation appears required for efficient signal transduction and antigen presentation. Studies in this aim will examine whether changes in dimer-forming ability, evaluated as rotational and energy transfer changes measurements, correlate with the efficacy of antigen presentation.