Allosteric intermediates in hemoglobin .1. Nanosecond time-resolved circular dichroism spectroscopy
Allosteric intermediates in hemoglobin .1. Nanosecond time-resolved circular dichroism spectroscopy
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DOI：
10.1021/bi952247s
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发表时间：
1996-07-02
期刊：
影响因子：
2.9
通讯作者：
Kliger, DS
中科院分区：
文献类型：
--
作者：
Bjorling, SC;Goldbeck, RA;Kliger, DS
对光解血红蛋白-CO 复合物 (HbCO) 进行的时间分辨圆二色性 (TRCD) 研究可探测 Hb 中的室温蛋白质弛豫，包括 R --> T 变构转变。近紫外 (250-400 nm) 光谱区光解中间体的 TRCD 光谱通过四级结构对芳香族残基 UV CD 的影响，对 α(1) beta(2) 界面处的 T 类结构进行诊断。紫外和索雷区中基于卟啉的跃迁的 TRCD，反映了血红素和芳香族残基之间的跃迁偶极子耦合，其半径足够宽以允许血红素界面和二聚体间形成相互作用，受到光解中间体的三级和四级结构的调节。在 Hb 协同性的变构核心模型中，Fe-CO 键断裂引发血红素结构变化，被认为是血红素穹顶，并通过 F 螺旋传递到 alpha(1) beta(2) 界面。 TRCD 结果根据时间分辨吸收研究的动力学信息进行分析，提出了随后的三级和四级构象变化通过蛋白质传播的机制的具体特征。特别是，UV-TRCD 表明 α(1) beta(2) 界面在几百纳秒内通过从 R 界面转变为 T 样界面来响应血红素的初始事件。在平衡 T 态 deoxyHb 出现之前数十微秒，α(1) beta(2) 界面处出现类似 T 的特征，这表明光解 HbCO 中的 R --> T 转变是一个逐步过程，正如之前时间分辨共振拉曼研究所表明的那样。
Time-resolved circular dichroism (TRCD) studies performed on photolyzed hemoglobin-CO complex (HbCO) probe room temperature protein relaxations in Hb, including the R --> T allosteric transition. TRCD spectroscopy of photolysis intermediates in the near-UV (250-400 nm) spectral region provides a diagnostic for T-like structure at the alpha(1) beta(2) interface via the effect of quaternary structure on the UV CD of aromatic residues, The TRCD of porphyrin-based transitions in the UV and Sorer regions, reflecting transition-dipole couplings between hemes and aromatic residues over a radius wide enough to permit heme-interface and inter-dimer interactions, is modulated by the tertiary and quaternary structure of photolysis intermediates. In the allosteric core model of Hb cooperativity, Fe-CO bond breakage initiates a heme structural change, thought to be heme doming, that is transmitted to the alpha(1) beta(2) interface via the F helix. The TRCD results, analyzed in light of kinetic information from time-resolved absorption studies, suggest specific features for the mechanism by which the ensuing tertiary and quaternary conformational changes propagate through the protein, In particular, the UV-TRCD indicates that the alpha(1) beta(2) interface responds within several hundred nanoseconds toinitial events at the heme by shifting from an R toward a T-like interface. The appearance of T-like character at the alpha(1) beta(2) interface tens of microseconds before the appearance of equilibrated T state deoxyHb indicates that the R --> T transition in photolyzed HbCO is a stepwise process, as previously suggested by time-resolved resonance Raman studies.