EPR STUDIES OF MEMBRANE PROTEINS

阴离子交换通道(带3)是一个95K的跨膜 人类红细胞中的一种蛋白质，它有助于 HCO3对氯的重要生理交换。这个 带3的细胞质结构域提供一个膜结合部位 糖酵解酶和血红蛋白以及附着部位 用于将膜骨架连接到脂质双层。这些 后一种功能表明，频段3充当组织中心 对于蛋白质-蛋白质相互作用来说是至关重要的 保持双凹面的形状，并到不寻常的 红细胞的粘弹性特性。的长期目标 拟议的研究是为了表征结构特征 与其作为阴离子转运功能有关的带3 并提供对其更多的了解 通过对组织功能的动态特性的阐述来组织功能 特异性膜骨架-带3相互作用。 两种反应性双功能自旋标记试剂， 双)磺基琥珀酰亚胺(BSSDP)和 双(磺基琥珀酰亚胺)-5-羟基氮杂环己酸酯(BSSDA)，已被 它允许带3在以下位置选择性地交联 在其胞外阴离子结合区或在一个 细胞外单体-单体接触面完好 红血球。现在将使用蛋白质测序来确定 带3上的氨基酸残基是通过 在两个位置都有自旋标记探针。这些东西的空间排列 将对功能和结构上重要的地点进行调查 通过电子顺磁共振(EPR)波谱 分析探头-探头相互作用的线形。这些是相同的 自旋标记将用于饱和转移电子顺磁共振(ST-EPR) 3带自转运动的量化测量 完好的红细胞和鬼膜制剂 各种生理条件。这些研究的目的是 阐明特定相互作用的动态特性 以旋转运动为基础的膜骨架蛋白和带3 对相互作用的性质和程度的探索。这些 研究还将扩大到包括表现出 形状和脆性异常导致溶血性贫血 膜骨架成员之间的相互作用改变 和/或其与带3的连接。互补ST-EPR研究 将在马来酰亚胺自旋标记的锚蛋白和蛋白质上进行 4.2结合到红细胞膜上，以定义 带3胞质结构域之间接触的灵活性 和下面的膜骨架。

The anion exchange channel (band 3) is a 95 k transmembrane protein in the human erythrocyte which facilitates the physiologically important exchange of HCO3 for Cl. The cytoplasmic domain of band 3 provides a membrane binding site for glycolytic enzymes and hemoglobin as well as attachment site for bridging the membrane skeleton to the lipid bilayer. These latter functions suggest that band 3 acts as an organizing center for protein-protein interactions which are crucial to the maintenance of the biconcave shape and to the unusual viscoelastic properties of the erythrocyte. The long term goals of the proposed research are to characterize structural features of band 3 which are relevant to its function as an anion transport protein and to provide an increased understanding of its organizing functions through elucidation of dynamic properties of specific membrane skeleton-band 3 interactions. Two reactively bifunctional spin label reagents, bis)sulfosuccinimidyl)-4-doxylpimelate (BSSDP) and bis(sulfosuccinimidyl)-5-doxylazelate (BSSDA), have been developed which allow selective cross-linking of band 3 at residues in its extracellular anion binding domain or at an extracellular monomer-monomer contact surface in intact erythrocytes. Protein sequencing will now be employed to define the amino acid residues on band 3 which are cross-linked by the spin label probes at both sites. The spatial arrangement of these functionally and structurally important sites will be investigated by electron paramagnetic resonance (EPR) spectroscopy by analyzing line shapes for probe-probe interactions. These same spin labels will be employed in saturation transfer EPR (ST-EPR) measurements for quantitating the rotational motion of band 3 in intact erythrocytes and ghost membrane preparations under a variety of physiological conditions. The aim of these studies is to elucidate dynamic properties of specific interactions between membrane skeletal proteins and band 3 using rotational motion as a probe for the nature and extent of the interactions. These studies will also be extended to include erythrocytes exhibiting abnormal shape and fragility resulting in hemolytic anemia due to altered interactions between members of the membrane skeleton and/or its attachment to band 3. Complementary ST-EPR studies will be carried out on maleimide spin labeled ankyrin and protein 4.2 bound to erythrocyte ghost membranes in oder to define the flexibility of contact between the cytoplasmic domain of band 3 and the underlying membrane skeleton.