Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy
Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy
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DOI：
10.1073/pnas.0709760104
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发表时间：
2007-12-04
影响因子：
11.1
通讯作者：
Fayer, Michael D.
中科院分区：
文献类型：
--
作者：
Ishikawa, Haruto;Kim, Seongheun;Fayer, Michael D.
据了解，分子内二硫键在调节蛋白质稳定性和活性中发挥作用。由于二硫键共价连接蛋白质的不同成分，因此它们影响蛋白质结构。然而，二硫键对快速（亚皮秒至约 100 ps）蛋白质平衡结构波动的影响尚未通过实验得到表征。在这里，超快 2D-IR 振动回波光谱用于检查分子内二硫键对蛋白质神经球蛋白 (Ngb) 结构波动的限制。 Ngb 是在脊椎动物大脑中发现的一种球蛋白家族蛋白，可可逆地结合氧气。与肌红蛋白 (Mb) 一样，Ngb 具有经典的珠蛋白折叠，并且血红素周围的关键残基是保守的。此外，Mb (Mb-CO) 和 Ngb (Ngb-CO) 的血红素连接 CO 振动光谱实际上是相同的。然而，与 Mb 不同，人 Ngb 具有分子内二硫键，影响其氧亲和力和蛋白质稳定性。通过使用 2D-IR 振动回波光谱，我们通过观察有或没有二硫键的 CO 光谱扩散（2D-IR 线形状的时间依赖性）来研究 Ngb-CO 的平衡蛋白质动力学。尽管有和没有二硫键的 Ngb-CO 的线性 FTIR 光谱相似，但 2D-IR 测量表明，二硫键的破坏加速了不同蛋白质构型的平衡采样。观察结果表明，Ngb 中的分子内二硫键充当快速蛋白质动力学的抑制剂，即使消除它不会在蛋白质结构中产生显着的构象变化。
Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to approximate to 100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure.