Severcan Feride, Haris Parvez I
Department of Biology, Middle East Technical University, 06531, Ankara, Turkey.
Biopolymers. 2003 Aug;69(4):440-7. doi: 10.1002/bip.10392.
Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.
猪柠檬酸合酶（PCS）可作为一种模型酶，以深入了解蛋白质热稳定性的结构基础。通过跟踪其酰胺I带成分的变化，详细监测了PCS中特定二级结构元件的热解折叠特性。我们的研究结果表明，PCS经历不可逆的热变性。详细分析表明，不同的二级结构呈现多步转变，在不同温度下有一个主要转变和一个次要转变，在相同温度（30℃）下有一个非常小的初始转变。温度诱导的（1）H-（2）H交换变化、α-螺旋结构吸光度的降低以及聚集结构吸光度的增加的曲线图都有一个共同的多步转变，次要转变以45℃为中心，主要转变在59℃左右。相比之下，一个暂定为环结构的带显示出相同的次要和主要转变，但温度较低（分别为39℃和52℃）。在39-45℃发生的转变与聚集结构的出现无关。这种转变可能反映了蛋白质三级结构的变化。然而，在较高温度（52-59℃）发生的最终转变反映了多肽链的解折叠和聚集。傅里叶变换红外（FTIR）分析表明，PCS有一个热不稳定区域，该区域首先解折叠，比蛋白质的主要解折叠温度低约7℃。我们认为，这反映了高度灵活的环段的解折叠，进而触发了PCS主要为螺旋的核心结构的解折叠。
