Song Xiang-Jin, Yuan Yue, Simplaceanu Virgil, Sahu Sarata Chandra, Ho Nancy T, Ho Chien
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Biochemistry. 2007 Jun 12;46(23):6795-803. doi: 10.1021/bi602654u. Epub 2007 May 12.
Model-free-based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy and carbonmonoxy forms of chain-specific, isotopically (15N and 2H) labeled tetrameric hemoglobin (Hb) using 15N-relaxation parameters [longitudinal relaxation rate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34 degrees C) and two magnetic field strengths (11.7 and 14.1 T). In both deoxy and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, beta146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, alpha31Arg and beta123Thr, neighbors in the intradimer (alpha1beta1) interface, exhibit stiffening upon CO binding. These findings imply a role for alpha31Arg and beta123Thr in the intradimer communication but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intradimer (alpha1beta1) interface (i.e., B, G, and H helices and the GH corner) and possible involvement of several amino acid residues (e.g., alpha31Arg, beta3Leu, beta41Phe, beta123Thr, and beta146His) in the allosteric pathway. Several amino acid residues at the intradimer interfaces, such as beta109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.
基于无模型的核磁共振动力学研究，使用在两个温度（29和34摄氏度）以及两个磁场强度（11.7和14.1特斯拉）下测量的15N弛豫参数[纵向弛豫率（R1）、横向弛豫率（R2）和异核核Overhauser效应（NOE）]，对链特异性、同位素（15N和2H）标记的四聚体血红蛋白（Hb）的脱氧和碳氧形式的多肽主链酰胺N-H键向量进行了研究。在人类正常成人血红蛋白（Hb A）的脱氧和碳氧形式中，除了环区域和某些螺旋-螺旋连接外，大多数氨基酸残基的酰胺N-H键在快速时间尺度（纳秒到皮秒）上是刚性的。虽然在脱氧Hb A中是刚性的，但已发现β146His在CO形式中其主链运动不受限制，推测是由于其氢键/盐桥网络的断裂。我们现在有了Hb在溶液中这种结构转变的直接动力学证据。虽然在脱氧状态下非常灵活，但二聚体内（α1β1）界面中的邻居α31Arg和β123Thr在CO结合后表现出刚性增强。这些发现暗示了α31Arg和β123Thr在二聚体内通讯中的作用，但与X射线晶体学的结果相矛盾。我们还发现二聚体内（α1β1）界面（即B、G和H螺旋以及GH转角）有相当大的灵活性，并且几个氨基酸残基（例如α31Arg、β3Leu、β41Phe、β123Thr和β146His）可能参与变构途径。二聚体内界面处的几个氨基酸残基，如β109Val，似乎参与了可能的构象交换过程。本研究得出的动力学图景为基于X射线晶体学结果的Hb A协同氧合的立体化学机制的传统描述提供了新的见解。
