Santambrogio P, Levi S, Arosio P, Palagi L, Vecchio G, Lawson D M, Yewdall S J, Artymiuk P J, Harrison P M, Jappelli R
Department of Biomedical Science and Technology, University of Milano, San Raffaele Hospital, Italy.
J Biol Chem. 1992 Jul 15;267(20):14077-83.
Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.
人铁蛋白是一种多聚体铁储存蛋白，由两种亚基类型按不同比例组成：H链和L链。尽管已显示这两种基因产物与铁的反应性存在差异，但其生物学功能尚未阐明。基于铁蛋白典型的高稳定性可能与其铁储存功能相关这一假设，我们研究了在不同变性条件下H链和L链的铁蛋白同聚物。此外，我们分析了13种H链变体，这些变体在哺乳动物H链保守区域存在改变。在所有变性实验中，H链铁蛋白的稳定性均低于L链铁蛋白。在盐酸胍变性实验中差异更大，其最终产物是完全展开的肽，而在酸性变性实验中差异较小，其最终产物是具有类似于“熔球”性质的肽。对H链变体的研究表明：（i）铁蛋白稳定性不受壳层内表面或外表面暴露区域且不参与链内或链间相互作用的改变的影响；（ii）亚基间相互作用所涉及序列的改变会降低稳定性，如N端延伸的缺失或沿疏水和亲水通道的替换；（iii）四螺旋束内的2个氨基酸被同源L链的氨基酸替换会增加稳定性。其中一个残基参与L链中的盐桥形成，我们得出结论，H型和L型铁蛋白之间的稳定性差异在很大程度上归因于该盐桥对L亚基折叠的稳定作用。
