Heteropolymer Ferritins Structure-Function Studies

 描述(申请人提供)：铁蛋白是多亚单位铁储存和解毒蛋白，在铁的生物管理中发挥核心作用。在脊椎动物中，铁蛋白由H和L两种类型的24个亚基组成，它们以不同的比例共同组装，并具有组织特异性分布。均聚物H链含有铁氧合酶中心，分子氧或过氧化氢在该中心将Fe(II)快速氧化为Fe(III)。尽管来自不同生物的铁蛋白有许多共同的结构特征，但铁的吸收、氧化、沉积和动员的化学成分明显不同。已经分别对重组均聚物H链或重组L链铁蛋白进行了广泛的研究，但没有对杂多聚合物H/L铁蛋白进行研究。[令人惊讶的是，尽管脊椎动物组织中普遍存在不同H与L亚基比例的杂多铁蛋白(等铁蛋白)，但人们对这些蛋白质以及H和L亚基在铁吸收和矿化过程中所起的互补作用知之甚少。这项研究计划的目标是研究重组杂多铁蛋白(模拟体内自然产生的铁蛋白)中铁摄取和沉积的结构与功能关系，并表征导致遗传性铁病的两个致病L铁蛋白变体的稳定性和功能性。具体地说，我们计划研究(A)H和L亚基在铁氧化和矿化过程中的互补作用，并确定这一过程中的铁-蛋白质中间产物，(B)L链突变和铁含量对蛋白质热稳定性的影响，以及(C)铁蛋白空腔内形成的铁核的磁性和结晶度。为了实现这一点，结合了定点突变、pH值统计/血氧测定法、 将使用停流快速动力学技术、紫外-可见光谱和荧光光谱、差示扫描量热法和穆斯堡尔光谱。本文提出的实验应该有助于详细了解杂多铁蛋白中铁的沉积化学，以及H和L亚基的不同比例如何影响异铁蛋白的生化和功能性质。它还将提供对遗传性神经铁蛋白病症的生化过程的见解。此外，拟议的铁蛋白铁核穆斯堡尔测量应该为铁蛋白外壳内可控尺寸的纳米颗粒的结构、设计和开发提供重要的见解 广泛的应用范围，从电子到生物医学。

 DESCRIPTION (provided by applicant): Ferritins are multi-subunit iron storage and detoxification proteins that play a central role in the biological management of iron. In vertebrates, ferritins consist of 24 subunits of two types, H and L that co-assemble in various ratios with a tissue specific distribution. The homopolymer H- chain contains ferroxidase centers where the rapid oxidation of Fe(II) to Fe(III) occurs by either molecular oxygen or hydrogen peroxide. While ferritins from different organisms share many common structural features, the chemistries of iron uptake, oxidation, deposition and mobilization differ markedly. Extensive studies have been performed, separately, with either recombinant homopolymer H-chain or recombinant L-chain ferritin, but not with the heteropolymer H/L ferritin. [Surprisingly, and despite the widespread occurrence of heteropolymer ferritins of different H to L subunit ratio (isoferritins) in tissues of vertebrates], very little is known about these proteins and the complementary roles that H and L subunits play during iron uptake and mineralization. The goals of this research proposal are to investigate the structure-function relationships of iron uptake and deposition in recombinant heteropolymer ferritins, [which mimic naturally occurring ferritins in-vivo], and to characterize the stability and functionality of two pathogenic L-ferriti variants responsible for a hereditary ferritinopathy disorder. Specifically, we plan to study (a) te complementary roles of H and L subunits in iron oxidation and mineralization and identify iron-protein intermediates during this process, (b) the effect of L-chain mutations and iron content on the protein thermostability and (c) the magnetism and crystallinity of the iron core formed inside the ferritin cavity. To achieve this, a combination of site-directed mutagenesis, pH stat/oximetry, stopped-flow rapid kinetics techniques, UV- visible and fluorescence spectroscopy, differential scanning calorimetry and Mössbauer spectroscopy will be employed. The experiments proposed here should lead to a detailed understanding of the chemistry of iron deposition in heteropolymer ferritins and how different proportions of H and L subunits affect the biochemistry and functional properties of isoferritins. It will also provide insights into the biochemical processes responsible for the hereditary neuroferritinopathy disorder. Additionally, the proposed Mössbauer measurements of ferritin iron core should provide important insights into the structure, design, and development of controlled size nanoparticles within the ferritin shell for a broad range of applications ranging from electronics to biomedicine.