Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high pressure FTIR spectroscopy
Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high pressure FTIR spectroscopy
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高压 FTIR 光谱揭示天然和 de novo Cro 蛋白单体变体之间结构稳定性和协同性的差异
作者：
H. Imamura;Y. Isogai;M. Kato
人们普遍认为压力会影响蛋白质的结构和动力学;然而，潜在的机制仍然没有得到解决。我们以前的研究已经使用模型肽研究了压力对基本二级结构元素的影响，因为这些肽代表了理解压力对更复杂结构影响的基础。本研究针对天然存在的噬菌体λ Cro（天然Cro）和从头设计的λ Cro（SN 4 m）的单体变体，它们是α + β蛋白。SN 4 m与天然Cro的序列有75%的差异，但结构几乎相同。因此，这些蛋白质的折叠特性的比较是有意义的。压力和温度可变的傅里叶变换红外光谱分析表明，天然Cro的α-螺旋和β-折叠通过压力和温度协同可逆地展开，而SN 4 m的α-螺旋和β-折叠不通过压力协同展开;即，SN 4 m的α-螺旋在比β-折叠高得多的压力下展开，并且随着温度的升高不可逆地展开。SN 4 m的α-螺旋的较高解折叠压力表明存在SN 4 m的中间结构，其不保留β-折叠结构但保留α-螺旋。这些结果表明，天然Cro的α-螺旋是通过α-螺旋和β-折叠之间的整体三级接触来稳定的，而SN 4 m的α-螺旋是通过α-螺旋之间的局部三级接触来稳定的。
It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.