BINDING SITE IN VITAMIN D-BINDING PROTEIN

维生素 D 结合蛋白中的结合位点

• 批准号: 2143725
• 负责人: RAHUL RAY
• 金额: $ 14.03万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143725
• 关键词: 25 hydroxycholecalciferol; affinity labeling; blood proteins; carbene; chemical binding; chemical structure; high performance liquid chromatography; hormone binding protein; mass spectrometry; molecular site; nuclear magnetic resonance spectroscopy; peptides; photoactivation; protein sequence; protein structure function; vitamin D; vitamin analog

Vitamin D binding protein (DBP), a major serum protein, binds to vitamin D and its metabolites with high affinity, and in turn, transports them to target organs and tissues, leading to the observed calciotropic properties of vitamin D hormone. Our objectives are to probe the vitamin D sterol-binding domain of DBP by our recently developed photoaffinity labelling method. Thus, we plan to photoaffinity label purified human serum DBP (hDBP) with 3H-25-ANE, a radiolabelled photoaffinity analog of 25-hydroxyvitamin D3, followed by proteolytic and chemical cleavages, and isolation of the labelled peptide/peptides. Mass spectrometric analysis of this peptide fragment/fragments will enable us to define the vitamin D-binding site in hDBP. We will also develop additional photoaffinity analogs of 25-OH-D3 containing (i) photoactive groups at various part of the parent molecule, (ii) spacer arm having different lengths, and (iii) photo probes with carbene precursors; and carry out studies similar to those described with 3H-25-ANE. This will lead to unequivocal identification of the vitamin D sterol-binding domain in hDBP as well as points of contact within the binding site.

维生素 D 结合蛋白 (DBP) 是一种主要的血清蛋白，与维生素结合 D 及其代谢物具有高亲和力，进而将它们转运至 靶器官和组织，导致观察到的促钙 维生素 D 激素的特性。 我们的目标是探索维生素 通过我们最近开发的光亲和力获得 DBP 的 D 甾醇结合结构域 标记方法。 因此，我们计划光亲和标记纯化的人类 血清 DBP (hDBP) 与 3H-25-ANE，一种放射性标记的光亲和类似物 25-羟基维生素 D3，然后进行蛋白水解和化学切割，以及 标记肽的分离。 质谱分析 这个肽片段将使我们能够定义维生素 hDBP 中的 D 结合位点。 我们还将开发额外的光亲和力 25-OH-D3 的类似物在不同部分含有 (i) 光活性基团 母体分子，(ii) 具有不同长度的间隔臂，以及 (iii) 具有卡宾前体的光探针；并进行类似的研究 那些用3H-25-ANE描述的。 这将导致明确的 hDBP 中维生素 D 甾醇结合域的鉴定以及 结合位点内的接触点。