GIANT HEMOGLOBIN LINKER CHAIN L1 DISULFIDE CONNECTIVITY

巨型血红蛋白连接链 L1 二硫键连接

• 批准号: 7355040
• 负责人: AUSTEN F RIGGS
• 金额: $ 0.12万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355040
• 关键词: GIANT; HEMOGLOBIN; LINKER; CHAIN; L1

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The extracellular hemoglobin of annelids and tube worms are multisubunit proteins of up to 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having O2 binding chains and "linker" chains. The linker chain L1 of the hemoglobin of Lumbricus terrestris contains a 38-39 residue segment with a repeating pattern of cysteinyl residues (CysX6)3 -CysX5-CysX10-Cys. This pattern, not present in any globin sequence, corresponds exactly tao the cysteine rich repeats of the ligand binding domain of the low density lipoprotein (LDL) receptor of man and Xenopus laevis. The disulfide connectivity of these domains has not been determined in the LDL receptor. The determination of the disulfide bonds in the linker chains is therefore an important first step in the understanding of the interaction between apolipoprotein E and the LDL receptor. Linker chain L1 was digested with a number of enzymes and the resulting peptide mixtures were a nalysed by matrix-assisted laser desorption mass spectrometry. Due to the huge number of possible disulfide bridges, the assignment of peaks to disulfide bridges containing peptides was extremely difficult. Therefore HPLC fractionation of peptide mixtures was carried out. For the identification of disulfide containing peptides fractions of interest further enzymatic and chemical reactions are performed. Identification of the resulting peptide products provides information that is useful for mapping the disulfide linkages. Ultimately, we found that MALDI-ITMS of the enzyme digested protein provided the highest quality mapping data. A paper describing the technique and discussing the results is in press.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。环节动物和管虫的细胞外血红蛋白是由多达 200 个多肽组成的多亚基蛋白，分子量至少为 3,900 kDa。它们与所有其他 Hb 的不同之处在于具有 O2 结合链和“连接”链。 Lumbricus terrestris 血红蛋白的连接链 L1 包含 38-39 个残基片段，其重复模式为半胱氨酰残基 (CysX6)3 -CysX5-CysX10-Cys。这种模式不存在于任何球蛋白序列中，与人和非洲爪蟾的低密度脂蛋白（LDL）受体的配体结合结构域的富含半胱氨酸的重复序列完全对应。 LDL 受体中这些结构域的二硫键连接尚未确定。 The determination of the disulfide bonds in the linker chains is therefore an important first step in the understanding of the interaction between apolipoprotein E and the LDL receptor.连接链 L1 用多种酶消化，所得肽混合物通过基质辅助激光解吸质谱法进行分析。由于存在大量可能的二硫桥，将峰分配给含有肽的二硫桥非常困难。因此，对肽混合物进行了 HPLC 分级分离。为了鉴定含有二硫键的感兴趣的肽级分，进行了进一步的酶促和化学反应。所得肽产物的鉴定提供了可用于绘制二硫键图谱的信息。最终，我们发现酶消化蛋白质的 MALDI-ITMS 提供了最高质量的图谱数据。一篇描述该技术并讨论结果的论文正在出版。