Luger K, Szadkowski H, Kirschner K
Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland.
Protein Eng. 1990 Mar;3(4):249-58. doi: 10.1093/protein/3.4.249.
Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold beta alpha barrel protein, was modified to produce a 10-fold beta alpha homologue in Escherichia coli. It contained a duplicate of the two C-terminal beta alpha units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (beta alpha)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (beta alpha)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (beta alpha)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type (beta alpha)8 protein. The data suggest that the folding of the (beta alpha)10 variant is controlled thermodynamically both in vivo and in vitro.
在两端都含有二级结构冗余片段的蛋白质序列，在折叠成野生型三级结构的几种可能的环状排列变体方面具有先验选择。为了验证这一假设，对来自酵母的磷酸核糖邻氨基苯甲酸异构酶基因进行了修饰，该基因是一种单结构域8倍β-α桶状蛋白，在大肠杆菌中产生了一种10倍β-α同源物。它在其N端融合了超二级结构的两个C端β-α单元的重复序列。通过溶解在氯化胍溶液中并复性，大部分蛋白质从破碎细胞的不溶性部分中回收。从可溶性部分中纯化出纯净的蛋白质。纯化后的（β-α）10蛋白几乎具有完全的酶活性。两种蛋白质的吸光度、荧光和圆二色光谱以及可逆的解折叠行为也与原始的（β-α）8蛋白的性质非常相似。用内肽酶消化将纯净的和复性后的（β-α）10变体都转化为具有野生型（β-α）8蛋白N端序列和分子量的相同大片段。数据表明，（β-α）10变体的折叠在体内和体外都受到热力学控制。
