Structural dynamism and analysis of functional domain of membrane proteins in signal transduction.

(1)采用共振镜检测方法（IAsys^<TM>）进行体外蛋白结合试验，在红细胞蛋白4.1 （4.1 r ^<80>）的nh_2末端30kDa结构域内鉴定出两个不同的钙调蛋白（CaM）结合位点：Ca^<2+>敏感位点和Ca^<2+>不敏感位点。在Ca^<2+>存在和不存在的情况下，CaM以1:1的化学计量量结合到4.1R^<80>。Ca^<2+>和CaM调节4.1R^<80>与膜蛋白、糖蛋白C （Glycophorin C， GPC）、Band 3和p55的相互作用。这种调节需要CaM结合到4.1R^<80>的Ca^<2+>敏感位点和-不敏感位点。(2)关于4.1R^<80>与GPC或p55相互作用的分子基础，以及4.1R^<80>在调节构成GPC-4.1R^<80>-p55三元配合物的各种蛋白质-蛋白质相互作用中的作用，我们知之甚少。我们发现4.1R^<80>使p55与GPC结合的亲和力增加了一个数量级，这意味着4.1R^<80>调节了p55与GPC之间的相互作用。(3)确定了4.1R^<80>的30kDa结构域的晶体结构，呈三叶草状结构。三叶草的每一个裂片都包含一个特定的结合位点，用于带3、GPC或p55。在分子的中心区域，靠近三个裂片连接的地方是两个独立的CaM结合区。(4) 4.1R的另一种异构体135kDa 4.1R （4.1R^<135>）在红母细胞中表达。4.1R^<135>在30kDa结构域nh_2末端的上游有一个额外的209个氨基酸的多肽，称为头片（HP）。我们的结果支持4.1R^<135>的HP可能调节30kDa结构域与红母细胞膜的结合。(5) 4.1G蛋白是4.1R的亚型，在红母细胞中也有表达。我们的实验支持4.1G与4.1R^<135>的膜结合特性不同，提示4.1G和4.1R^<135>可能在红母细胞中具有不同的功能。

(1)In vitro protein binding assays by the resonant mirror detection method (IAsys^<TM>) identified two distinct calmodulin (CaM) binding sites within the NH_2-terminal 30kDa domain of erythrocyte protein 4.1 (4.1R^<80>) : a Ca^<2+>-sensitive and -insensitive binding sites. CaM bound to 4.1R^<80> at a stoichiometry of 1:1 both in the presence and absence of Ca^<2+>. Ca^<2+> and CaM regulated interactions of 4.1R^<80> with membrane proteins, Glycophorin C (GPC), Band 3 and p55. This regulation required binding of CaM to both Ca^<2+>-sensitive and -insensitive sites in 4.1R^<80>.(2)Little is known regarding the molecular basis for the interaction of 4.1R^<80> with either GPC or p55 and regarding the role of 4.1R^<80> in regulating the various protein-protein interactions that constitute the GPC-4.1R^<80>-p55 ternary complex. We showed that 4.1R^<80> increases the affinity of p55 binding to GPC by an order of magnitude, implying that4.1R^<80> modulates the interaction between p55 and GPC.(3)The crystal structure of 30kDa domain of 4.1R^<80> has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for Band 3, GPC or p55. At a central region of the molecule near where the three lobes are joined are two separate CaM binding regions.(4)Another isoform of 4.1R, 135kDa 4.1R (4.1R^<135>) is expressed in erythroblasts. 4.1R^<135> has an additional 209 amino acids polypeptide, referred to as head-piece (HP), upstream of the NH_2-terminal end of the 30kDa domain. Our results support that HP in 4.1R^<135> may regulate the 30kDa domain binding to membrane in erythroblasts.(5)The protein 4.1G, which is an isoform of 4.1R, is also expressed in erythroblasts. Our experiments support that the membrane binding properties of 4.1G are different from those of 4.1R^<135>, suggesting that 4.1G and 4.1R^<135> may share different functions in erythroblasts.