MacColl R, Kapoor S, Montellese D R, Kukadia S, Eisele L E
Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA.
Biochemistry. 1996 Dec 3;35(48):15436-9. doi: 10.1021/bi961334x.
At 45 degrees C, phycocyanin 645 maximally undergoes a reversible and stable conformational change. The change is observed in the visible (chromophore) region of the absorption and circular dichroism (CD) spectra. In the absorption spectrum, the absorbance is lower at 45 degrees C but remains much closer to the normal spectrum than to a strongly denatured spectrum. In the CD, a similar situation exists except that a negative band on the blue edge of the spectrum is much more strongly affected at 45 degrees C than the other bands. On returning to 20 degrees C, all these changes are restored to the original states. The protein is an alpha 2 beta 2 dimer at both 20 and 45 degrees C, and CD in the far-UV shows the identical protein secondary structures at both 20 and 45 degrees C. Fluorescence studies show that energy transfer occurs at both temperatures. At 50 degrees C the results are saliently different as the secondary structure changes and the spectral changes are mostly irreversible. At 50 degrees C, some monomers (alpha beta) are produced, and these monomers are very unstable at that temperature, resulting in the formation of some fully denatured polypeptides. Stable monomers can be produced at 20 degrees C and have visible absorption and CD spectra identical to the dimer at 45 degrees C. Therefore, the chromophores are reporting a tertiary conformational change at 45 degrees C, in which the two halves of the dimer each assume a monomer-like conformation prior to dissociating. These results are compared with a hypothesis for the chromophore topography, and the CD change at the blue edge of the spectra may result from the separation at 45 degrees C of a chromophore pair.
在45摄氏度时，藻蓝蛋白645会最大程度地发生可逆且稳定的构象变化。这种变化在吸收光谱和圆二色性（CD）光谱的可见光（发色团）区域可以观察到。在吸收光谱中，45摄氏度时的吸光度较低，但仍比强变性光谱更接近正常光谱。在CD光谱中，情况类似，只是光谱蓝边的负带在45摄氏度时比其他带受到的影响更大。回到20摄氏度时，所有这些变化都会恢复到原始状态。该蛋白质在20摄氏度和45摄氏度时均为α2β2二聚体，远紫外区的CD显示在20摄氏度和45摄氏度时蛋白质二级结构相同。荧光研究表明，在这两个温度下都会发生能量转移。在50摄氏度时，结果明显不同，因为二级结构发生变化且光谱变化大多不可逆。在50摄氏度时，会产生一些单体（αβ），这些单体在该温度下非常不稳定，导致形成一些完全变性的多肽。在20摄氏度时可以产生稳定的单体，其可见吸收光谱和CD光谱与45摄氏度时的二聚体相同。因此，发色团报告了45摄氏度时的三级构象变化，其中二聚体的两半在解离前各自呈现类似单体的构象。将这些结果与发色团拓扑结构的假设进行了比较，光谱蓝边的CD变化可能是由于45摄氏度时一对发色团的分离所致。
