MOSSBAUER STUDIES OF LOW-SPIN FERRIC PROTEINS

低自旋三价铁蛋白的莫斯鲍尔研究

• 批准号: 3438502
• 负责人: CHARLES E SCHULZ
• 金额: $ 5.67万
• 依托单位: KNOX COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1990-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438502
• 关键词: Mossbauer spectrometry; acidity /alkalinity; cytochrome b; electron spin resonance spectroscopy; heme; hemoprotein; hemoprotein structure; ionic strengths; iron; ligands; metalloproteins; myoglobin; peroxidases; solvents; temperature

The physical characterization of the iron site in low-spin ferric heme proteins is very important in working toward an understanding of the structure-function relationships in these systems. The proposed research will investigate two aspects of the protein structure: i) how the ligand field of the iron is perturbed by slight changes in the amino acid sequence and structure of the protein, and ii) the temperature dependence of the spin-lattice relaxation and how it varies with changes in the solvent. The first line of investigation is made possible by the expression of a synthetic rat hepatic cytochrome b5 gene in E. coli, where it is expressed in extremely large quantities, up to 20% of total cell protein. Specific modifications can be made in the cytochrome by cartridge mutagenesis. These modified proteins will be studied by EPR and Mossbauer spectroscopy, and the spectra will be parametrized by a S=1/2 Spin Hamiltonian, which can then be used as the basis for a ligand field characterization of the iron site. The temperature dependence of the Mossbauer spectra will serve as a further test of the fractal model for protein vibrational modes. EPR measurements of various heme and iron-sulfur proteins indicate that the phonon density of states in these systems is consistent with a model in which the protein has a fractional dimension of 1.1-1.8. These measurements were made for temperatures below 12K. Mossbauer spectroscopy is an ideal method to extend these studies to the temperature range 15K- 200K because 1) the integrated intensity of the Mossbauer spectrum is not as strong a function of temperature as is EPR; and, 2) EPR measurements indicate that the fluctuation rates for these temperatures should be in the range to which Mossbauer spectra are sensitive. We propose to first study myoglobin azide and the effects of the solvent on its fractional dimension, with the work later to be extended to the Horseradish peroxidase cyanide complex, cytochrome b5, and small heme model compounds.

低自旋三价铁中铁位的物理性质 血红素蛋白质是非常重要的， 了解这些结构与功能的关系 系统. 拟议的研究将调查两个方面， 蛋白质结构：i）铁的配体场如何 受氨基酸序列轻微变化的干扰， 蛋白质的结构，和ii）温度依赖性 自旋-晶格弛豫以及它如何随着 溶剂后 调查的第一线是由 合成的大鼠肝细胞色素b5基因在E. 在大肠杆菌中，它以极大的量表达， 20%的细胞蛋白。 具体修改可以在 通过盒式诱变获得细胞色素。 这些改性 蛋白质将通过EPR和穆斯堡尔光谱进行研究， 光谱将由S=1/2自旋哈密顿量参数化， 它可以作为配体场的基础 铁网站的特点。 穆斯堡尔谱的温度依赖性将有助于 作为对蛋白质振动分形模型的进一步检验， modes. 各种血红素和铁硫的EPR测量 蛋白质表明，在这些声子态密度 系统与蛋白质具有 分数维为1.1-1.8。 这些测量是在 温度低于12 K。 穆斯堡尔光谱学是一种理想的 方法，以扩大这些研究的温度范围15 K- 200 K，因为1）穆斯堡尔的积分强度 光谱不像EPR那样是温度的强函数; EPR测量结果表明， 这些温度应该在穆斯堡尔谱 光谱是敏感的。 我们建议首先研究肌红蛋白叠氮化物 以及溶剂对其分数维的影响， 这项工作后来扩展到辣根过氧化物酶 氰化物复合物、细胞色素b5和小血红素模型 化合物.