Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: Time-resolved UV resonance Raman evidence for intra-dimer coupling
Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: Time-resolved UV resonance Raman evidence for intra-dimer coupling
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DOI：
10.1016/j.jmb.2004.05.013
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发表时间：
2004-07-16
影响因子：
5.6
通讯作者：
Spiro, TG
中科院分区：
文献类型：
--
作者：
Balakrishnan, G;Tsai, CH;Spiro, TG
通过定点诱变、时间分辨吸收和紫外共振拉曼 (UVRR) 光谱研究了消除血红蛋白 (Hb) 四聚体中特定叔氢键的动力学效应。 Trpalpha14... Thralpha67 和 Trpbeta15... Serbeta72 氢键连接 α 链和 β 链中的 A 和 E 螺旋，并建议在 HbCO 光脱氧后断裂最早的蛋白质中间体（R-脱氧），以及涉及酪氨酸残基的第二对氢键。受体残基 Thralpha67 和 Serbeta72 突变为 Val 和 Ala 可消除 A-E H 键，但已显示对配体结合亲和力或协同性或 T 态四元相互作用的光谱标记没有显着影响。然而，这些突变对 R-脱氧中间体的特性以及导致 T 状态的后续步骤的动力学具有深远且意想不到的影响。初始四元接触（R-T 中间体）的形成加速了一个数量级，但 T 状态的锁定延迟了 2 倍。这些速率效应对于突变或双突变基本上是相同的，表明上二聚体表现为机械耦合动力单元。 Rdeoxy UVRR 光谱提供了二聚体内偶联的进一步证据，其中一个或两个突变消除了酪氨酸差异强度，尽管只有色氨酸 H 键受到直接影响。概述了机械耦合的可能机制，涉及通过 alpha(1)beta(1)（和 alpha(2)beta(2) 界面传递力。目前的观察结果表明，四元运动可以在大约 100 ns 的时间尺度上发生。他们还表明，完整的螺旋间氢键实际上减慢了 Hb 中的初始四元运动，但加速了 T 接触的锁定。(C) 2004由爱思唯尔有限公司出版
The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trpalpha14... Thralpha67 and Trpbeta15... Serbeta72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (R-deoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thralpha67 and Serbeta72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the R-deoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (R-T intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the up dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1), (and alpha(2)beta(2) interface. The present observations establish that quaternary motions can occur on the similar to100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts. (C) 2004 Published by Elsevier Ltd.