Greenfield N J, Hitchcock-DeGregori S E
Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Protein Sci. 1993 Aug;2(8):1263-73. doi: 10.1002/pro.5560020809.
Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.
圆二色性被用于研究αα-原肌球蛋白和AcTM43（一种由43个残基组成的肽，被设计作为原肌球蛋白N端结构域的模型）的折叠情况。该肽的序列为AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC。在具有生理离子强度和pH值的缓冲液中，该肽在低温（<25℃）下似乎形成了卷曲螺旋结构。然而，该肽的折叠和去折叠过程是非协同的。当将圆二色光谱作为温度的函数进行检测时，在222、208和280nm处跟踪椭圆率时，表观折叠程度有所不同。光谱的去卷积表明，在远紫外区至少有三条成分曲线对圆二色性有贡献。一条成分曲线类似于天然αα-原肌球蛋白卷曲螺旋α-螺旋的圆二色光谱。第二条曲线类似于球状蛋白质中发现的单链短α-螺旋片段的光谱。第三条曲线类似于无规卷曲构象的多肽的光谱。这些结果表明，随着该肽的折叠，在大多数卷曲螺旋形成之前，α-螺旋含量增加。当将横纹肌αα-原肌球蛋白的圆二色光谱作为温度的函数进行检测时，其去折叠也不是完全协同的。当温度从0℃升高到25℃时，卷曲螺旋减少，传统α-螺旋型光谱增加，没有形成无规卷曲。主要转变发生在40℃，是一个协同转变，其特征是所有剩余的卷曲螺旋消失，同时无规卷曲增加。
