Gong Qingguo, Simplaceanu Virgil, Lukin Jonathan A, Giovannelli Janel L, Ho Nancy T, Ho Chien
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Biochemistry. 2006 Apr 25;45(16):5140-8. doi: 10.1021/bi052424h.
We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights regarding the roles of allosteric effectors in regulating the structure and function of hemoglobin. The classical two-state/two-structure allosteric mechanism for the cooperative oxygenation of hemoglobin cannot account for the structural and functional properties of this protein and needs to be revised.
我们应用剩余偶极耦合（RDC）方法，以拉伸聚丙烯酰胺凝胶作为排列介质，研究了碳氧形式的、用（2）H和（15）N标记的人类正常成人重组血红蛋白（rHb A）以及低氧亲和力突变重组血红蛋白rHb(α96Val→Trp)在不存在和存在变构效应剂肌醇六磷酸（IHP）时的溶液四级结构。我们最近的RDC结果[Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517 - 520]表明，溶液中HbCO A的四级结构是两个先前确定的晶体结构R（在高盐条件下生长的晶体）和R2（在低盐条件下生长的晶体）之间的动态集合。基于对T、R和R2结构几何坐标的比较，有人提出Hb A的氧合遵循从T到R再到R2的转变途径，其中R是中间结构[Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113 - 11117]。这里给出的结果表明，IHP可以使HbCO A的溶液四级结构略微向R结构偏移。在不存在IHP时，rHbCO(α96Val→Trp)的溶液四级结构与存在IHP时HbCO A的结构相似，这与rHbCO(α96Val→Trp)对氧的亲和力低于Hb A一致。此外，IHP在使rHbCO(α96Val→Trp)的溶液四级结构向R结构和向T结构偏移方面具有更强的作用，这与IHP导致其氧亲和力更显著降低一致。这项工作中给出的结果以及最近文献中报道的其他结果清楚地表明，血红蛋白的结合形式存在多种四级结构。这些结果也为变构效应剂在调节血红蛋白结构和功能中的作用提供了新的见解。经典的血红蛋白协同氧合的两态/两结构变构机制无法解释该蛋白质的结构和功能特性，需要进行修正。
