15N-NMR STUDIES OF RHODOSPIRILLUM RUBRUM CYTOCHROME C2

血红素蛋白的结构和功能一直是一个研究热点。 调查了几十年，因为他们参与了大多数 基本的生物过程，如氧气运输、氧化 磷酸化和光合作用。大量的细胞色素， 携带一个电子的血红素蛋白，是已知的参与 电子传输链是最厌氧的有机体。 在这些蛋白质中，细胞色素C构成了一个具有很强 结构相似，但有几个例外，但性质截然不同。 这个实验室的长期目标是确定结构如何 不同的细胞色素允许它们在他们的 各自的系统。这种特异性既涉及氧化还原的匹配 与系统中其他电子载体的电势和 它们之间结合相互作用的专一性。 这个实验室的另一个兴趣是开发15N-核磁共振作为一种工具，用于 对小蛋白质的研究。细胞色素C是大小适中的蛋白质 哪些氮原子在结构和结构中扮演着异常重要的角色 蛋白质的表面电荷分布。15N可以合并 利用生物合成技术转化红色红螺菌细胞色素c2 为生物体提供15NH4离子。均匀浓缩的细胞色素 将通过现有的程序进行提纯，其N-核磁共振谱检测如下 PH、温度和氧化状态的函数。共鸣将是 使用已知的氨基酸移位尽可能具体地分配 分组和双共振技术。~(15)N的化学位移变化 与pH的共振将与已知发生在 通过氧化还原电位的变化检测到的、可见的分子 吸收光谱以确定哪些基团能够改变这些 属性。分辨的15N共振的运动的时间尺度可以 可以从1H-15N核超室效应的值中估算出来。这 信息将被用于确定不同的刚性 分子的结构元素。表面的局部运动 赖氨酸残基可能会在未来的实验中用来绘制出 细胞色素与其氧化还原伙伴之间的相互作用。

The structure and function of heme proteins has been an area of intense investigation for decades because of their involvement in the most fundamental biological processes such as oxygen transport, oxidative phosphorylation and photosynthesis. A large number of cytochromes, one-electron carrying heme proteins, are known that are involved in electron transport chains of all that the most anaerobic of organisms. Among these proteins, the cytochromes C constitute a group having very similar structure, with a few exceptions, but very different properties. It is a long range goal of this laboratory to determine how the structure of different cytochromes allow them to function optimally in their respective systems. The specificity involves both the matching of redox potentials to those of the other electron carriers in the system and the specificity of the binding interactions between them. Another interest of this laboratory is to develop 15N-NMR as a tool for the study of small proteins. Cytochromes C are appropriately sized proteins in which nitrogen atoms play unusually important roles in the structure and surface charge distribution of the protein. 15N can be incorporated biosynthetically into the cytochrome c2 of Rhodospirillum rubrum by supplying the organism with 15NH4 ion. The uniformly enriched cytochrome will be purified by existing procedures and its N-NMR spectrum examined as a function of pH, temperature and oxidation state. Resonance will be assigned as specifically as possible using known shifts of amino acid groups and double resonance techniques. Changes in chemical shifts of 15N resonances with pH will be correlated with ionizations known to occur in the molecule that are detected by changes in redox potential and visible absorption spectrum to determine which groups are capable of altering these properties. The time scale of the motions of resolved 15N resonances can be estimated from the value of the 1H-15N nuclear Overhouser effect. This information will be used to determine the rigidity of the different structural elements of the molecule. The local motion of the surface lysine residues might be used in future experiments to map out the site of interaction between the cytochrome and its redox partners.