Buelt M K, Bernlohr D A
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1990 Aug 14;29(32):7408-13. doi: 10.1021/bi00484a008.
The adipocyte lipid binding protein (ALBP) is a member of a multigene family of low molecular weight proteins which stoichiometrically and saturably bind hydrophobic ligands and presumably facilitate intracellular lipid metabolism. To probe the structure-function relationship of the binding domain of ALBP, chemical modification has been employed. Modification of the two cysteinyl residues of ALBP (Cys1 and Cys117) with a variety of sulfhydryl reagents decreased the apparent affinity for oleic acid in the following order of effectiveness: methyl methanethiosulfonate much much less than p-(chloromercuri)benzenesulfonic acid less than N-ethylmaleimide (NEM) = 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Thiol titration of ALBP with DTNB in the presence of bound oleate resulted in the modification of a single cysteinyl residue. The oleate-protected cysteine was identified as Cys117 by modification with a combination of reversible (DTNB) and irreversible (NEM) sulfhydryl reagents in the presence or absence of saturating oleic acid. Cys117-NEM ALBP exhibited a large decrease in binding affinity while Cys1-NEM ALBP exhibited normal binding properties. Neither the modification of ALBP with NEM nor the addition of oleic acid had a significant effect on protein structure, as judged by circular dichroic analysis. These results suggest that Cys117 of ALBP resides in the ligand binding domain and that site-specific modification can be utilized to assess the conformational flexibility of the binding cavity.
脂肪细胞脂质结合蛋白（ALBP）是一个低分子量蛋白质多基因家族的成员，它能以化学计量比且饱和地结合疏水配体，并可能促进细胞内脂质代谢。为了探究ALBP结合结构域的结构 - 功能关系，已采用化学修饰方法。用多种巯基试剂对ALBP的两个半胱氨酸残基（Cys1和Cys117）进行修饰，降低了对油酸的表观亲和力，其有效性顺序如下：甲硫基磺酸甲酯远小于对（氯汞基）苯磺酸小于N - 乙基马来酰亚胺（NEM）= 5,5'-二硫代双（2 - 硝基苯甲酸）（DTNB）。在结合油酸的情况下，用DTNB对ALBP进行巯基滴定导致单个半胱氨酸残基被修饰。通过在存在或不存在饱和油酸的情况下，用可逆（DTNB）和不可逆（NEM）巯基试剂组合进行修饰，将油酸保护的半胱氨酸鉴定为Cys117。Cys117 - NEM ALBP的结合亲和力大幅下降，而Cys1 - NEM ALBP表现出正常的结合特性。通过圆二色性分析判断，用NEM对ALBP进行修饰或添加油酸对蛋白质结构均无显著影响。这些结果表明，ALBP的Cys117位于配体结合结构域中，并且位点特异性修饰可用于评估结合腔的构象灵活性。
