Glasgow B J, Abduragimov A R, Yusifov T N, Gasymov O K, Horwitz J, Hubbell W L, Faull K F
Department of Pathology, UCLA School of Medicine, Los Angeles, California 90095, USA.
Biochemistry. 1998 Feb 24;37(8):2215-25. doi: 10.1021/bi9720888.
Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of proteins that carry lipids. Amino acid sequences for two of the six isolated isoforms were assigned by a comparison of molecular mass measurements with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentatively sequence assigned using the same criteria. The most abundant isoform has a measured mass of 17 446.3 Da, consistent with residues 19-176 of the putative precursor (calculated mass 17 445.8 Da). Chemical derivatization of native and reduced/denatured protein confirmed the presence of a single intramolecular disulfide bond in the native protein. Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide bridge. Mass measurements of tryptic fragments identified C119 as the free cysteine and showed that the single intramolecular disulfide bond joined residues C79 and C171. Circular dichroism indicated that tear lipocalins have a predominant beta-pleated sheet structure (44%) that is essentially retained after reduction of the disulfide bond. Circular dichroism in the far-UV showed reduced molecular asymmetry and enhanced urea-induced unfolding with disulfide reduction indicative of relaxation of protein structure. Circular dichroism in the near-UV shows that the disulfide bond contributes to the asymmetry of aromatic sites. The effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restricts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformational changes that involves aromatic residues. The disulfide bridge in tear lipocalins is important in conferring protein rigidity and influencing ligand affinity. The disulfide bond appears highly conserved so that these findings may have implications for the entire lipocalin superfamily.
已确定泪液视黄醇结合蛋白（一种携带脂质的新型蛋白质家族成员）中二硫键基序的结构和功能特征。通过将分子量测量值与根据cDNA预测的蛋白质序列和可用的N端蛋白质序列数据计算出的质量进行比较，确定了六个分离的同工型中的两个的氨基酸序列。使用相同标准对第三个同工型进行了初步序列分配。最丰富的同工型测得质量为17446.3Da，与推定前体的19 - 176位残基一致（计算质量为17445.8Da）。天然蛋白和还原/变性蛋白的化学衍生化证实天然蛋白中存在单个分子内二硫键。天然、还原和变性蛋白与4 - 吡啶二硫化物和二硫代双（2 - 硝基苯甲酸）的反应性表明，完整的二硫桥显著限制了对游离半胱氨酸的接触。胰蛋白酶片段的质量测量确定C119为游离半胱氨酸，并表明单个分子内二硫键连接C79和C171残基。圆二色性表明泪液视黄醇结合蛋白具有主要的β - 折叠结构（44%），在二硫键还原后基本保留。远紫外区的圆二色性显示分子不对称性降低，尿素诱导的解折叠增强，二硫键还原表明蛋白质结构松弛。近紫外区的圆二色性表明二硫键有助于芳香族位点的不对称性。使用结合视黄醇的固有光学活性监测二硫键还原对配体结合的影响。完整的二硫键降低了泪液视黄醇结合蛋白对视黄醇的亲和力，并限制了视黄醇对天然脂质的置换。二硫键还原伴随着涉及芳香族残基的配体诱导构象变化的显著改变。泪液视黄醇结合蛋白中的二硫桥在赋予蛋白质刚性和影响配体亲和力方面很重要。二硫键似乎高度保守，因此这些发现可能对整个视黄醇结合蛋白超家族有影响。
