Electron Spin Relaxation in Model Membranes

描述(申请人提供)：这项工作旨在加深对作为重要生物学过程基础或与健康疾病有关的模型膜和生物膜、生物活性多肽和膜蛋白的动态分子性质以及结构、功能和关联的了解，并将使用最新的一维和二维电子自旋共振(ESR)技术来更好地解决这些问题。具体项目包括：研究活的RBL-2H3(和其他相关细胞)和质膜小泡(PMV)质膜的动态结构域结构，以将结构域结构的变化与抗原激活后IgE受体的信号联系起来，以验证受体激活受周围脂类结构调控的假说。这项研究将基于最近的结果，这些结果表明存在液体有序和液体无序的磁区。将研究来自流感病毒血凝素(WT20)的融合肽和曲率诱导蛋白对膜有序性的影响，以检验基于WT20增加脂类头部基团有序性的观察结果，双层有序性增加与更稳健的膜融合相关的假设，以及膜曲率诱导蛋白将导致负电荷脂类头部基团有序性的变化。借助于FRED基团培育的强大的脉冲偶极ESR波谱(PDS)来研究膜蛋白的结构和聚集，将确定自旋标记的WT20肽的聚集数和结构随WT20浓度的变化，如结构变化将与膜中脂质有序性的变化相关联。利用多频ESR和PDS技术，在已有方法和结果的基础上，对合成的Walp和KALP多肽进行了跨膜螺旋倾斜和多肽聚集等疏水双分子层厚度与多肽长度不匹配的研究。PDS的其他研究将用于确定大分子膜蛋白及其复合体的结构。这包括自旋标记的含有BAR结构域的蛋白质，以确定膜结合时发生的构象变化。第二个研究是人类突触核蛋白的膜结合构象，包括他们的帕金森病(PD)连锁突变体，以检验我们的假设，即α-突触核蛋白(AS)可能在体内以延伸螺旋和U形形式存在，每一种形式都已在模型系统中得到证实。第三项研究是确定完整的化学感受器的结构和它们在激活时所经历的构象变化。重点将放在四个蛋白质单位CHEA/CHEW与受体形成的复合体上。这些研究将涉及与领先研究小组的广泛合作。可能的临床应用包括检测免疫反应过程中的细胞膜变化，防止病毒进入，以及神经系统疾病(包括帕金森病)。与公共卫生相关：我们正在研究蛋白质和细胞膜的结构，以及蛋白质与细胞膜的其他成分(如脂质和胆固醇)相互作用的方式。我们的研究将集中在了解细胞之间通过交换细胞成分进行交流的机制。蛋白质结构和细胞通讯方式的紊乱可能会导致过敏、动脉硬化、帕金森氏症和其他疾病。

DESCRIPTION (provided by applicant): This work is aimed at developing greater understanding of the dynamic molecular properties, as well as the structure, function, and association, of model and biological membranes, bioactive peptides, and membrane proteins that underlie important biological processes or are implicated in health disorders, and the latest one and two-dimensional electron-spin resonance (ESR) technologies will be employed to better address these issues. Specific projects include the following: The study of the dynamic domain structure of the plasma membrane in live RBL-2H3 (and other related cells) and plasma membrane vesicles (PMV) will be employed to correlate the change in domain structure with signaling by the IgE receptor after activation by antigen to test the hypothesis that receptor activation is modulated by the domain structure of the surrounding lipids. This study will be based on recent results, which showed the existence of Liquid-ordered and Liquid-disordered domains. The effects of fusion peptides, such as from hemagglutinin of influenza virus (wt20), and curvature- inducing proteins on membrane ordering will be studied to test the hypotheses that increased bilayer ordering is associated with more robust membrane fusion and that membrane curvature-inducing proteins will induce changes in the head group ordering of negatively charged lipids, based on the observation that wt20 increases the ordering of the lipid headgroups. By means of the powerful pulsed-dipolar ESR spectroscopy (PDS) cultivated by the Freed group for studying membrane protein structure and aggregation, the aggregation number and structural changes of spin-labeled wt20 peptide as a function of wt20 concentration will be determined, such as structural changes will be correlated with the changes in lipid ordering profile in the membrane. By means of multi-frequency ESR and PDS, the effects of hydrophobic mismatch between peptide length and lipid bilayer thickness, such as tilting of trans-membrane helices and peptide aggregation, will be studied for synthetic WALP and KALP peptides, based on previously developed methods and results. Additional studies by PDS will be directed to the determination of structures of large membrane proteins and their complexes. This includes spin-labeled BAR domain-containing proteins to determine the conformational changes that occur upon membrane binding. A second study is that of membrane-bound conformations of human synucleins including their Parkinson's disease (PD) linked-mutants to test our hypothesis that alpha- synuclein (aS) may exist in vivo in both extended helix and U-shaped form, each of which have already been demonstrated in model systems. A third study is to determine the structure of intact chemoreceptors and the conformational changes they undergo upon activation. The focus will be on the complex that the four protein unit, CheA/CheW, forms with the receptor. These studies will involve extensive collaborations with leading research groups. Possible clinical applications include detection of membrane changes during immune response, prevention of viral entry, an neurological disorders (including PD). PUBLIC HEALTH RELEVANCE: We are studying structures of proteins and cell membranes, as well as the way proteins interact with other components of cell membranes, such as lipids and cholesterols. Our study will focus on understanding mechanisms by which cells communicate with each other through exchange of cellular components. Disorder in the structure of the proteins and the manner of cell communication may lead to allergic conditions, arteriosclerosis, Parkinson's and other diseases.