Highly asymmetric interactions between globin chains during hemoglobin assembly revealed by electrospray ionization mass spectrometry
Highly asymmetric interactions between globin chains during hemoglobin assembly revealed by electrospray ionization mass spectrometry
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DOI：
10.1021/bi034035y
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发表时间：
2003-08-26
期刊：
影响因子：
2.9
通讯作者：
Kaltashov, IA
中科院分区：
文献类型：
--
作者：
Griffith, WP;Kaltashov, IA
采用电喷雾质谱和圆二色性光谱研究了牛血红蛋白在溶液中的组装动力学。对应于各种蛋白质物种（四聚体，二聚体，血红素缺乏的二聚体，以及载脂蛋白和holo单体）的离子信号的强度被用来估计这些物种在溶液中的相对分数作为pH值的函数。折叠蛋白质的分数为每个观察到的物种估计基于相应的离子物种在质谱中的电荷状态分布。累积数（平均在整个蛋白质人口）与圆二色性数据在Soret带和远紫外区，分别在良好的协议。质谱数据证实，血红蛋白解离涉及血红素首先从β-二聚体的β-链中丢失以形成血红素缺乏的二聚体物质的步骤。该二聚体进一步解离以产生全α链和脱辅基α链。前者在中性pH下紧密折叠成相对紧凑的结构，而后者总是表现出显着的骨架无序。将蛋白质溶液酸化至pH 4导致部分血红素解离，并且还显著增加α-链中的主链柔性。血红素在pH低于4时从α-链上完全解离与二聚体和四聚体血红蛋白物质从质谱中完全消失相一致。实验数据提供了强有力的证据表明，部分非结构化的脱辅基-β-链与紧密折叠的全-α-链结合形成血红素缺陷二聚体是血红蛋白组装的初始步骤。这种结合将β-链锁定在高度有序的构象中，这允许有效的血红素获取，然后对接两个血红蛋白二聚体以形成蛋白质的四聚体形式。两条链在组装过程中的作用的不对称性是令人惊讶的，因为它们具有相当高的序列同源性（约200）。43%），并强调内在蛋白质紊乱的功能重要性。该研究还表明了质谱作为一种分析工具的巨大潜力，能够阐明高度异质性系统中的蛋白质相互作用机制。
Dynamics of bovine hemoglobin assembly was investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. Intensities of ionic signals corresponding to various protein species (tetramers, dimers, heme-deficient dimers, as well as apo- and holo-monomers) were used to estimate relative fractions of these species in solution as a function of pH. The fraction of folded protein for each observed species was estimated based on charge-state distributions of corresponding ionic species in the mass spectra. The cumulative numbers (averaged across the entire protein population) were in good agreement with circular dichroism data at the Soret band and in the far-UV region, respectively. The mass spectral data confirm that hemoglobin dissociation involves a step where heme is first lost from the beta-chain of the alphabeta-dimer to form a heme-deficient dimeric species. This dimer dissociates further to produce a holo-alpha-chain and an apo-alpha-chain. The former is tightly folded into a comparatively compact structure at neutral pH, while the latter always exhibits significant backbone disorder. Acidification of the protein solution to pH 4 leads to partial heme dissociation and significant increase of the backbone flexibility in the alpha-chains as well. Complete dissociation of the heme from the alpha-chains at a pH below 4 coincides with the total disappearance of the dimeric and tetrameric hemoglobin species from the mass spectra. The experimental data provide strong evidence that binding of a partially unstructured apo-beta-chain to a tightly folded holo-alpha-chain to form a heme-deficient dimer is the initial step of hemoglobin assembly. Such binding locks the beta-chain in a highly ordered conformation, which allows for an efficient heme acquisition, followed by docking of two hemoglobin dimers to form a tetrameric form of the protein. The asymmetry of the roles of the two chains in the assembly process is surprising, given a rather high sequence homology (ca. 43%) and highlights functional importance of intrinsic protein disorder. The study also demonstrates a tremendous potential of mass spectrometry as an analytical tool capable of elucidating protein interaction mechanisms in highly heterogeneous systems.