Sahu Sarata C, Simplaceanu Virgil, Gong Qingguo, Ho Nancy T, Glushka John G, Prestegard James H, Ho Chien
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
J Am Chem Soc. 2006 May 17;128(19):6290-1. doi: 10.1021/ja060023z.
Human normal adult hemoglobin (Hb A) is a tetrameric protein molecule of ~64 kDa consisting of two identical -chains and two identical -chains of 141 and 146 amino acid residues each and four bound heme moieties. In the oxygen-free form of Hb A, also known as deoxyhemoglobin A (deoxy-Hb A), the hemes are paramagnetic with S = 2. We have measured the one-bond spin-spin couplings (1JNH + 1DNH) on (15N,2H)-labeled deoxy-Hb A in solution as a function of magnetic field strengths from 11.7 to 21.1 T and found that these couplings are linearly proportional to the square of the magnetic field. This field dependence provides an opportunity to extract the residual dipolar couplings (RDCs, 1DNH) and, thus, to compare predictions about the solution structure of deoxy-Hb A to crystal structures for this molecule. Such comparison is essential for our understanding of the structure, dynamics, and function of this allosteric protein under conditions close to the physiological state. This report illustrates the usefulness of using the magnetic-field dependent RDCs to determine the solution structure of a large paramagnetic protein. This method is especially valuable for those proteins whose structures must be determined in an oxygen-free environment.
人类正常成人血红蛋白（Hb A）是一种约64 kDa的四聚体蛋白质分子，由两条相同的α链和两条相同的β链组成，每条链分别含有141和146个氨基酸残基，以及四个结合的血红素部分。在Hb A的无氧形式中，也称为脱氧血红蛋白A（deoxy-Hb A），血红素具有顺磁性，S = 2。我们测量了溶液中（15N,2H）标记的脱氧-Hb A上的一键自旋-自旋耦合（1JNH + 1DNH）作为磁场强度从11.7到21.1 T的函数，发现这些耦合与磁场的平方呈线性比例关系。这种场依赖性提供了一个机会来提取残余偶极耦合（RDCs，1DNH），从而将关于脱氧-Hb A溶液结构的预测与该分子的晶体结构进行比较。这种比较对于我们理解这种变构蛋白在接近生理状态的条件下其结构、动力学和功能至关重要。本报告说明了使用磁场依赖性RDCs来确定大型顺磁性蛋白质溶液结构的有用性。这种方法对于那些必须在无氧环境中确定其结构的蛋白质特别有价值。
