Griffith Wendell P, Kaltashov Igor A
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Biochemistry. 2003 Aug 26;42(33):10024-33. doi: 10.1021/bi034035y.
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 alpha beta-dimer to form a heme-deficient dimeric species. This dimer dissociates further to produce a holo-alpha-chain and an apo-beta-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.
通过电喷雾电离质谱法和圆二色光谱法监测溶液中的单体/寡聚体平衡，研究了牛血红蛋白组装的动力学。对应于各种蛋白质种类（四聚体、二聚体、血红素缺陷二聚体以及脱辅基和全蛋白单体）的离子信号强度用于估计这些种类在溶液中的相对比例随pH的变化。根据质谱中相应离子种类的电荷态分布，估计每个观察到的种类的折叠蛋白比例。累积数量（在整个蛋白质群体中平均）分别与Soret带和远紫外区域的圆二色数据高度一致。质谱数据证实，血红蛋白解离涉及一个步骤，即血红素首先从αβ - 二聚体的β链上丢失，形成血红素缺陷的二聚体种类。这种二聚体进一步解离产生一个全蛋白α链和一个脱辅基β链。前者在中性pH下紧密折叠成相对紧凑的结构，而后者总是表现出明显的主链无序。将蛋白质溶液酸化至pH 4会导致部分血红素解离，同时α链中的主链柔韧性也显著增加。在pH低于4时，血红素从α链上完全解离与质谱中二聚体和四聚体血红蛋白种类的完全消失相吻合。实验数据提供了有力证据，表明部分无结构的脱辅基β链与紧密折叠的全蛋白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，这允许有效地获取血红素，随后两个血红蛋白二聚体对接形成蛋白质的四聚体形式。考虑到相当高的序列同源性（约43%），两条链在组装过程中作用的不对称性令人惊讶，并突出了内在蛋白质无序的功能重要性。该研究还证明了质谱作为一种分析工具在阐明高度异质系统中蛋白质相互作用机制方面的巨大潜力。
