Falconi M, Desideri A, Cupane A, Leone M, Ciccotti G, Peterson E S, Friedman J M, Gambacurta A, Ascoli F
Department of Biology and INFM, University of Rome "Tor Vergata," 00133 Roma, Italy.
Biophys J. 1998 Nov;75(5):2489-503. doi: 10.1016/S0006-3495(98)77693-3.
Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.
对不等齿毛蚶同二聚体血红蛋白的一个突变体进行了分子动力学模拟、低温可见吸收光谱和共振拉曼光谱研究，该突变体在亚基界面处的苏氨酸72被异亮氨酸取代。分子动力学模拟表明，在Thr-72→Ile突变体中，一些已被证明在配体结合中起作用的残基围绕着与天然血红蛋白CO衍生物的x射线结构中观察到的方向和距离相似的方向和距离波动，尽管整体结构仍处于T态。可见吸收光谱数据表明，在脱氧形式下，突变体中的Soret带比天然蛋白质中的不对称性更小，这表明血红素结构更平面；此外，这些数据表明，在突变体蛋白质的配体结合态和非配体结合态中，血红素与溶剂的相互作用相似，这与在天然蛋白质中观察到的情况不同。脱氧突变体蛋白质的“构象敏感”带III相对于天然蛋白质向低能方向移动了>100 cm-1，约为两种蛋白质低温光产物中观察到的移动量的一半，这表明血红素环境的极性较小或疏水性更强。共振拉曼光谱数据显示，脱氧突变体相对于天然蛋白质，铁近端组氨酸拉伸模式向低频方向有轻微移动，这可以用模拟中观察到的Phe-97苯环堆积变化或血红素口袋中水分子的丢失来解释。与后一种解释一致的是，分子动力学模拟计算得出，动态进入亚基间界面的水分子数量在突变体中比在天然蛋白质中少。一氧化碳突变体衍生物的10 ns光产物的铁近端组氨酸拉伸频率比天然蛋白质高。这表明在突变体中，亚纳秒级的弛豫过程减慢，这与R结构的稳定一致。综上所述，分子动力学和光谱数据表明，Thr-72→Ile突变体表现出的较高氧亲和力主要是由于二聚体界面的局部扰动传播到血红素区域，扰乱了血红素环境的极性和丙酸酯相互作用。这些变化与突变体相对于天然蛋白质中T态的不稳定和R态的稳定一致。
