2D NMR STUDIES OF STRUCTURE & DYNAMICS OF CYTOCHROME C

在先前的工作中，使用二维质子NMR方法来确认， 正确的，或重新分配血红素的质子共振和超过一半的 马铁细胞色素C中的氨基酸 还有一些残留物 被分配到其他细胞色素C。 一个完整的任务， 相关蛋白质将为研究 蛋白质结构和动力学。 这也将提供一个特殊的 用于测试和使用新兴NMR技术的系统。 初步结果 这表明，先前被认为严重阻碍 将这些方法应用于较大的蛋白质（例如自旋扩散， T1和T2效应）实际上不是问题。 因此我们建议 应用这些已被证明是成功的分配策略， 马酶的归属 这里的成功将提供关键， 快速进一步指定其他亚铁细胞色素C种类（5种 提出）和它们的氧化对应物。 将使用NMR技术 定义局部二级结构和动力学以及 氧化还原状态和氨基酸取代的变化。 结构将 从NOE和耦合常数的时间依赖性确定。 覆盖亚纳秒和秒之间的范围的结构动力学 将被研究。 大部分运动信息将来自于 NOESY实验中的弛豫数据和化学交换效应。 总的来说，这些结果预计将提供有趣的见解， 蛋白质结构-功能关系以及蛋白质结构 动力学

In prior work two dimensional proton NMR methods were used to confirm, correct, or assign de novo the proton resonances of the heme and over half the amino acids in horse ferrocytochrome c. Also a number of residues have been assigned in other cytochromes c. A complete assignment of these related proteins would provide an unparalleled resource for studies of protein structure and dynamics. This would also provide an exceptional system for testing and using emerging NMR techniques. Preliminary results indicate that several effects earlier thought to pose serious barriers to the application of these methods to larger proteins (e.g. spin diffusion, T1 and T2 effects) are in fact not a problem. We therefore propose to apply these assignment strategies, already proven successful, to complete the assignment of the horse enzyme. Success here will provide the key to the rapid further assignment of additional ferrocytochrome c species (five are proposed) and their oxidized counterparts. NMR techniques will be used to define local secondary structure and dynamics and the effects thereon of change in redox state and amino acid substitution. Structure will be determined from the time dependence of the NOE and coupling constants. Structural dyamics covering the range between subnanoseconds and seconds will be studied. The majority of motional information will be derived from relaxation data and chemical exchange effects in the NOESY experiment. Overall these results are expected to provide interesting insights into protein structure-function relationships as well as protein structural dynamics.