Scholz Christian, Eckert Barbara, Hagn Franz, Schaarschmidt Peter, Balbach Jochen, Schmid Franz Xaver
Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
Biochemistry. 2006 Jan 10;45(1):20-33. doi: 10.1021/bi051922n.
SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.
SlyD是一种来自大肠杆菌胞质溶胶的假定折叠辅助蛋白，它由一个FKBP型的N端脯氨酰异构酶结构域和一个可能无结构的C端尾巴组成。我们制备了不含该尾巴的截短版本（SlyD），用于大肠杆菌的SlyD以及来自鼠疫耶尔森菌、梅毒螺旋体、多杀巴斯德菌和霍乱弧菌的SlyD直系同源物。它们在溶液中呈单体形式且可逆展开。所有SlyD变体都能以非常高的效率催化核糖核酸酶T1的脯氨酸限制型重折叠，特异性常数（kcat/KM）约为10(6) M(-1) s(-1)。这些大数值源于SlyD直系同源物对未折叠的RCM-T1的高亲和力，这体现在约1 microM的低KM值上。SlyD还表现出显著的伴侣蛋白特性。永久未折叠的蛋白质与SlyD高亲和力结合，从而抑制其脯氨酰异构酶活性。未折叠的蛋白质链不需要含有脯氨酸残基就能被SlyD识别和结合。SlyD家族中脯氨酰异构酶活性和伴侣蛋白特性的保守性表明，这些蛋白质可能在细菌胞质溶胶中充当真正的折叠辅助蛋白。SlyD蛋白也非常适合生物技术应用。作为融合伙伴，它们有助于重折叠并增加易聚集蛋白（如HIV-1的gp41胞外域片段）的溶解度。
