Electron Spin Relaxation in Model Membranes

描述（申请人提供）：这项工作的目的是发展动态分子特性，以及结构，功能和协会，模型和生物膜，生物活性肽和膜蛋白的基础重要的生物过程或涉及健康疾病，最新的一维和二维电子自旋共振（ESR）技术将被用来更好地解决这些问题。具体项目包括以下内容：在活RBL-2 H3（和其他相关细胞）和质膜囊泡（PMV）的质膜的动态域结构的研究将被用来关联域结构的变化与信号转导的IgE受体激活后，由抗原来测试的假设，受体激活是由周围脂质的域结构调制。这项研究将基于最近的结果，这表明存在液体有序和液体无序域。将研究融合肽（例如来自流感病毒血凝素（wt 20）的融合肽）和曲率诱导蛋白对膜有序化的影响，以检验以下假设：增加的双层有序化与更稳健的膜融合相关，以及膜曲率诱导蛋白将诱导带负电荷的脂质的头基有序化的变化，基于WT 20增加脂质头部基团的有序性的观察。通过Freed小组培养的用于研究膜蛋白结构和聚集的强大脉冲偶极ESR光谱（PDS），将确定自旋标记的wt 20肽的聚集数和结构变化作为wt 20浓度的函数，例如结构变化将与膜中脂质有序分布的变化相关。通过多频ESR和PDS，肽长度和脂质双层厚度之间的疏水性失配的影响，如跨膜螺旋的倾斜和肽聚集，将研究合成的WALP和KALP肽，基于先前开发的方法和结果。PDS的其他研究将致力于确定大膜蛋白及其复合物的结构。这包括自旋标记的含BAR结构域的蛋白质，以确定膜结合时发生的构象变化。第二项研究是人突触核蛋白的膜结合构象，包括其帕金森氏病（PD）连锁突变体，以测试我们的假设，即α-突触核蛋白（aS）可能以延长的螺旋和U形形式存在于体内，其中每一种都已经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与受体形成的复合物上。这些研究将涉及与主要研究小组的广泛合作。可能的临床应用包括检测免疫反应期间的膜变化、预防病毒进入、神经系统疾病（包括PD）。公共卫生相关性：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理解细胞通过交换细胞成分相互交流的机制。蛋白质结构和细胞通讯方式的紊乱可能导致过敏性疾病、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.