EPR STUDIES OF MEMBRANE PROTEINS

阴离子交换通道（带3）是一个95 K跨膜 人红细胞中的蛋白质， 生理上重要的HCO 3交换Cl。 的 带3的胞质结构域提供膜结合位点 糖酵解酶和血红蛋白以及附着位点 用于将膜骨架桥接到脂质双层。 这些 后一种功能表明，波段3起着组织中心的作用 蛋白质间的相互作用， 保持双凹的形状和不寻常的 红细胞的粘弹性。 的长期目标 拟议的研究是表征结构特征， 谱带3与其作为阴离子运输的功能有关 蛋白质，并提供更多的了解其 组织功能，通过阐明的动态特性， 特异性膜结合子-带3相互作用。 两种反应性双功能自旋标记试剂， 双）磺基琥珀酰亚胺基）-4-羟庚二酸酯（BSSDP）和 双（磺基琥珀酰亚胺基）-5-doxylazelate（BSSDA），已被 开发了允许带3在 在其胞外阴离子结合结构域中或在其胞外阴离子结合结构域中的 细胞外单体-单体接触表面 红细胞 蛋白质测序将被用来确定 条带3上的氨基酸残基，其通过 自旋标记探针在两个网站。 这些的空间布局 将对功能和结构上重要的部位进行研究 通过电子顺磁共振（EPR）光谱， 分析探针-探针相互作用的线形。 这些相同 自旋标记将用于饱和转移EPR（ST-EPR） 用于量化带3的旋转运动的测量 完整红细胞和血影膜制备物， 各种生理条件。 这些研究的目的是 阐明特定相互作用的动力学特性， 膜骨架蛋白和带3使用旋转运动作为 一种探测相互作用的性质和程度的探测器。 这些 研究还将扩展到包括表现出 形状和脆性异常，导致溶血性贫血， 改变了膜骨架成员之间的相互作用 和/或其与带3的连接。 补充ST-EPR研究 将进行马来酰亚胺自旋标记锚蛋白和蛋白质 4.2与红细胞血影膜结合， 带3的胞质结构域之间接触的灵活性 和底层的膜骨架。

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.