N-TERMINAL ACETYLATION AND PROTONATION OF INDIVIDUAL HEMOGLOBIN SUBUNITS

单个血红蛋白亚基的 N 末端乙酰化和质子化

• 批准号: 7355131
• 负责人: JAMES M MANNING
• 金额: $ 0.25万
• 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/7355131
• 关键词: TERMINAL; ACETYLATION; PROTONATION; INDIVIDUAL; HEMOGLOBIN

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 presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the {alpha}- or the {beta}-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the {alpha}-subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer?dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N-terminals of the {beta}-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer?dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the {alpha}-subunit, the slope of the plot of the tetramer?dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the {alpha}-subunits is less extensive than that of the {beta}-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在人成人血红蛋白的{α}-或{β}-亚基的N-末端存在丙氨酸（Ala）或乙酰丝氨酸（AcSer）而不是正常的瓦尔残基，赋予该蛋白一些新的和意想不到的特征。质谱分析证实这些取代是正确的，并且它们是唯一的取代。圆二色谱研究表明没有全球蛋白质构象的变化，等电聚焦显示不存在杂质。丙氨酸的存在下，在N-末端的{α}-亚基的配体血红蛋白的结果显着增加的碱性（增加pKa值）和减少的强度，在变构四聚体的亚基相互作用？二聚体界面O2结合的协同性也降低。在{β}-亚基的N-末端的Ala取代不产生这些效应。在任一亚基的N末端的丝氨酸的取代导致其完全乙酰化（在表达过程中）和四聚体的强度大幅下降？二聚体变构界面当Ala或AcSer存在于{α}-亚基的N末端时，四聚体的图的斜率？作为pH函数的二聚体结合/解离常数降低了60%。有人建议，由于网络的相互作用涉及的N和C末端的{α}-亚基是不太广泛的比{β}-亚基配位的人血红蛋白破坏有可能有一个深刻的影响血红蛋白功能，如增加碱性，对四聚体强度的影响，和协同性。