MECHANISMS OF STABILIZATION OF B-HEMEPROTEINS

这一建议解决了两种类型的问题，有关血红素含 蛋白质：关于一级序列之间关系的一般问题 和高等教育的作用的具体问题， 辅基在决定的结构和稳定性， 原生材料含血红素的肝细胞膜亲水区 选择细胞色素b5（cyt b5）作为模型系统。 关于cyt b5的功能和结构已经知道很多了。然而， 辅基之间相互作用的相对重要性， 蛋白质基质尚未完全了解，其结构由 单独的多肽链还没有被详细表征。细胞色素b5 将进行系统的研究，旨在确定各种 交互. （a）细胞色素b5的脱辅基蛋白将通过循环详细研究。 二色性和质子核磁共振光谱。现有 二级结构的元素将被识别， 它们相互挤压;结构的稳定性将 通过改变外部条件进行测试;血红素结合的动力学， 将监测其构象结果。 （B）将用原卟啉IX重构载脂细胞b5，即 没有中心铁原子的辅基。结构数据将 在“des-Fe”蛋白上获得，以提取 蛋白质和原卟啉环。 （c）将产生单位点和双位点突变蛋白质， 用与野生型相同的方法探测载脂蛋白 脱辅基蛋白突变体将揭示选择的氨基酸的贡献 酸对脱辅基蛋白的结构及其稳定性的影响。 将（a）、（B）和（c）中收集的数据与 有关还原和氧化形式的现有相应信息 全蛋白因此，氨基酸序列，辅基， 和铁原子在固定天然全蛋白构象和确定 其热力学性质将被单独表征。的 这项工作的结论将适用于更好地了解 蛋白质的结构决定因素。

This proposal addresses two types of problems relevant to heme-containing proteins: general questions on the relationship between primary sequence and secondary and tertiary structure, and specific questions on the role of the prosthetic group in determining the structure and stability of the native material. The heme-containing hydrophilic domain of hepatic cytochrome b5 (cyt b5) is chosen as model system. Much is known about cyt b5 function and structure. Yet the nature and relative importance of the interactions between the prosthetic group and the protein matrix are not fully understood, and the structure dictated by the polypeptide chain alone has not been characterized in detail. Cyt b5 will be subjected to systematic studies aimed at defining the various interactions. (a)The apoprotein of cyt b5 will be investigated in detail by circular dichroic and proton nuclear magnetic resonance spectroscopy. The existing elements of secondary structure will be identified and the way in which they pack against each other described; the stability of the structure will be tested by varying external conditions; the kinetics of heme binding and its conformational consequences will be monitored. (b)Apocyt b5 will be reconstituted with protoporphyrin IX, i.e. the prosthetic group without the central iron atom. Structural data will be obtained on the "des-Fe" protein to extract the interactions between the protein and the protoporphyrin ring. (c)Single- and double-site mutant proteins will be produced and their apoprotein probed by the same methods as applied to the wild-type apoprotein. The mutants will reveal the contribution of selected amino acids to the structure of the apoprotein and its stability. The data collected in (a), (b), and (c) will be compared to the corresponding information available on the reduced and oxidized forms of the holoprotein. Thus the roles of amino acid sequence, prosthetic group, and iron atom in fixing the native holoprotein conformation and determining its thermodynamic properties will be characterized individually. The conclusions of this work will apply to a better understanding of the structural determinants of proteins.