Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
J Phys Chem B. 2010 Dec 9;114(48):15958-68. doi: 10.1021/jp1044964. Epub 2010 Nov 15.
We used picosecond time-resolved fluorescence spectroscopy to characterize the fluorescence Stokes shift (FSS) response function of metal-free (or free-base, fbCytc) cytochrome c under the solution conditions that favor the native states of ferricytochrome c (FeCytc) and Zn(II)-substituted cytochrome c (ZnCytc). The intrinsic porphyrin chromophore serves in these experiments as a fluorescent probe of the structural fluctuations of the surrounding protein and solvent. Demetalation of the porphyrin destabilizes the folded structure of cytochrome c owing to the loss of the axial metal-histidine and metal-methionine bonds. Thus, these experiments examine how the time scales detected in a dynamic solvation experiment in a chromoprotein report changes in the character of motion. The FSS response function in fbCytc in water and pH 7 is well described by a biexponential response over the 100 ps to 50 ns regime with time constants of 1.4 and 9.1 ns; under similar conditions, ZnCytc exhibits a biexponential FSS response with time constants of 250 ps and 1.5 ns [Lampa-Pastirk and Beck, J. Phys. Chem. B 2004, 108, 16288]. These time constants correspond, respectively, to the correlation time scales for motions of the hydrophobic core and the solvent-contact layer of the protein. Both of the time constants observed in fbCytc are further lengthened upon addition of glycerol to the external solvent so that a significant fraction of the protein dynamics is rendered effectively static on the fluorescence time scale. The solvation reorganization energy, the time-integrated Stokes shift of the fluorescence spectrum, is reduced by about a third to 33 cm(-1) in 50% glycerol from 43 cm(-1) in water. These results are interpreted structurally using a model for Brownian diffusive motion with thermally activated barrier crossings on the protein-folding energy landscape. The results suggest that the mean-squared deviations of the structural fluctuations exhibited by fbCytc are nearly a factor of 10 larger than those of ZnCytc. This conclusion is consistent with the suggestion that fbCytc assumes a dynamic, partially unfolded structure with some of the characteristics of a molten globule.
我们使用皮秒时间分辨荧光光谱法来描述无金属（或自由碱基，fbCytc）细胞色素 c 在有利于天然状态的三价细胞色素 c（FeCytc）和锌取代细胞色素 c（ZnCytc）的溶液条件下的荧光斯托克斯位移（FSS）响应函数。在这些实验中，内在的卟啉发色团充当了周围蛋白质和溶剂结构波动的荧光探针。由于轴向金属-组氨酸和金属-蛋氨酸键的丧失，卟啉的脱金属会破坏细胞色素 c 的折叠结构。因此，这些实验研究了在色蛋白的动态溶剂化实验中检测到的时间尺度如何报告运动特征的变化。在水和 pH 7 下的 fbCytc 的 FSS 响应函数在 100 ps 至 50 ns 范围内很好地用双指数响应来描述，其时间常数为 1.4 和 9.1 ns；在类似的条件下，ZnCytc 表现出双指数 FSS 响应，其时间常数为 250 ps 和 1.5 ns [Lampa-Pastirk 和 Beck，J. Phys. Chem. B 2004，108，16288]。这些时间常数分别对应于疏水核心和蛋白质溶剂接触层的运动的相关时间尺度。在向外部溶剂中添加甘油后，fbCytc 中观察到的两个时间常数都进一步延长，以至于蛋白质动力学的很大一部分在荧光时间尺度上变得有效静态。溶剂重组能，即荧光光谱的时间积分斯托克斯位移，从水中的 43 cm(-1)降低到 50%甘油中的 33 cm(-1)，约减少了三分之一。这些结果从结构上使用布朗扩散运动模型和蛋白质折叠能面上的热激活势垒穿越进行解释。结果表明，fbCytc 表现出的结构波动的均方偏差几乎是 ZnCytc 的十倍。这一结论与 fbCytc 呈现出动态、部分展开结构的观点一致，其中具有一些无规卷曲的特征。
