Szilágyi A N, Kotova N V, Semisotnov G V, Vas M
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
Eur J Biochem. 2001 Mar;268(6):1851-60.
Refolding of pig muscle 3-phosphoglycerate kinase (PGK) from a mixture of its complementary proteolytic fragments that did not correspond to the individual domains resulted in a high degree of reactivation [Vas, M., Sinev, M.A., Kotova, N. & Semisotnov, G.V. (1990) Eur. J. Biochem. 189, 575--579]. An independent refolding of the 27.7 kDa C-terminal proteolytic fragment (which encompasses the whole C domain) has been noted, but the refolding ability of the 16.8-kDa N-terminal proteolytic fragment, which lacks a single subdomain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of initiation of refolding, pulse-proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This structure, however, remains unchanged upon further incubation of the N-terminal fragment, whereas refolding of the C-terminal fragment continues as detected by a further increase in proteolytic resistance. The non-native character of the folding intermediate of the N fragment is indicated by the elevated fluorescence intensity of the bound hydrophobic probe 8-anilino-1-naphtalene sulphonate. Its CD spectrum shows the formation of secondary structure distinct from the native one. The noncooperative phase-transition observed in microcalorimetry indicates the absence of a rigid tertiary structure, in contrast with the refolded C-terminal fragment for which a cooperative transition is seen. Size-exclusion chromatography supported the globular character of the intermediate, and showed its propensity to form dimers. No binding of the substrate, 3-phosphoglycerate (3-PGri), to the isolated N-terminal fragment, could be detected but the presence of the complementary C-terminal fragment led to restoration of the substrate binding ability of the N domain. Thus, refolding of the isolated N-terminal fragment yields a highly flexible, globular, potentially productive intermediate with non-native secondary structure and highly exposed hydrophobic clusters, which favour dimerization.
猪肌肉3 - 磷酸甘油酸激酶（PGK）由与其互补的蛋白水解片段混合复性而成，这些片段并不对应于单个结构域，复性后酶活性得到了高度恢复[瓦斯，M.，西涅夫，M.A.，科托娃，N.和谢米索特诺夫，G.V.（1990）《欧洲生物化学杂志》189卷，575 - 579页]。有人指出27.7 kDa的C端蛋白水解片段（包含整个C结构域）能够独立复性，但缺少N结构域中一个亚结构域的16.8 kDa N端蛋白水解片段的复性能力还有待观察。本文对分离片段的复性过程进行了比较。在复性开始的最初几秒内，脉冲蛋白水解实验表明两个片段都形成了具有中等蛋白酶抗性的结构。然而，进一步孵育N端片段时，该结构保持不变，而C端片段的复性则通过蛋白酶抗性的进一步增加得以检测到仍在继续。结合的疏水探针8 - 苯胺基 - 1 - 萘磺酸盐荧光强度升高，表明N片段折叠中间体具有非天然特性。其圆二色光谱显示形成了与天然结构不同的二级结构。微量量热法中观察到的非协同相变表明不存在刚性三级结构，这与复性后的C端片段形成协同转变形成对比。尺寸排阻色谱法支持中间体具有球状特征，并显示出其形成二聚体的倾向。未检测到底物3 - 磷酸甘油酸（3 - PGri）与分离的N端片段结合，但互补的C端片段的存在导致N结构域恢复了底物结合能力。因此，分离的N端片段复性产生了一种高度灵活、球状、具有潜在活性的中间体，其具有非天然二级结构和高度暴露的疏水簇，有利于二聚化。
