Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.
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机译:附睾视黄酸结合蛋白(ERABP)是附睾内腔中主要的雄激素依赖性蛋白,被认为与精子成熟有关。它与血清视黄醇结合蛋白(RBP)表现出高度的三维结构相似性。尽管两种蛋白都与类视黄醇相互作用,但RBP表现出广泛的特异性,以大致相等的亲和力结合视黄醇,视黄酸和视黄醛,而ERABP对全反式和9-顺式-视黄酸具有特异性。与此相一致的是,两种蛋白质的结合口袋是不同的:在RBP中,它主要是疏水的,而对于ERABP,它是两亲的,在结合口袋的开口端有一个带电残基的网络。为了研究这些带电残基的作用,已将Arg-80和Glu-63突变为异亮氨酸。产生的双重突变体Glu-63-> Ile / Arg-80-> Ile以及野生型蛋白随后在大肠杆菌中表达为融合蛋白,链霉亲和素识别序列(Strep)标记为他们的C总站。表达的蛋白可通过链霉亲和素亲和层析一步纯化,并使用荧光滴定法检查其配体结合特性。野生型ERABP仅结合视黄酸,而双突变体则能够以相似的亲和力结合视黄醇,视黄酸和视黄醛。这些观察结果为以下命题提供了实验依据:结合口袋开口端附近的带电残基负责限制ERABP对视黄酸的特异性。这些研究表明特异性改变可以被工程化为脂质运载蛋白。
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