Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Minami-josanjima-cho, Tokushima 770-8506, Japan.
BMC Biochem. 2010 Jul 24;11:27. doi: 10.1186/1471-2091-11-27.
A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heat-aggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N- or C-terminal truncated mutants and compared their properties with wild type AgsA.
AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23rd and C-terminal 11th amino acid (AA) from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50 degrees C. Destabilization of oligomeric complexes was observed for N-terminal 11- and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH) and citrate synthase (CS) but did not prevent the aggregation of insulin at 25 degrees C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25 degrees C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities), good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25 degrees C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25 degrees C.
Our data demonstrate that AgsA has several regions necessary for efficient chaperone activity: region(s) important for lysozyme chaperone activity are located outer surface of the oligomeric complex while those region(s) important for insulin are located inside the oligomeric complex and those for MDH are located within the N-terminal arm. In addition, the equilibrium between the oligomer and the dimer structures appears to be important for its efficient chaperone activity.
小热休克蛋白 AgsA 最初是从沙门氏菌肠炎血清型 Typhimurium 中分离出来的。我们之前证明 AgsA 是一种有效的伴侣蛋白，可减少大肠杆菌 rpoH 突变体中热聚集蛋白的数量。AgsA 似乎通过与体内其他伴侣蛋白合作来促进在致死温度下的存活。为了研究 AgsA 的聚集预防机制，我们构建了 N 端或 C 端截断突变体，并将其性质与野生型 AgsA 进行了比较。
AgsA 与小麦 sHsp16.9 具有显著的整体同源性，允许预测其三维结构。AgsA 的 N 端截断至第 23 位和 C 端截断至第 11 位氨基酸（AA）均保留了其体内伴侣活性。温度控制的凝胶过滤色谱显示，纯化的 AgsA 可在 50°C 下保持大的寡聚复合物。N 端 11- 和 17-AA 截断的 AgsA 的寡聚复合物不稳定；C 端 11-AA 截断的 AgsA 不能形成大的寡聚复合物。AgsA 可防止变性溶菌酶、苹果酸脱氢酶（MDH）和柠檬酸合酶（CS）的聚集，但在 25°C 时不能防止胰岛素的聚集。N 端 17-AA 截断的 AgsA 对 MDH 没有伴侣活性。C 端 11-AA 截断的 AgsA 对溶菌酶、MDH 和 CS 的伴侣活性较弱或没有，但在 25°C 时可防止胰岛素的聚集。当等量的 AgsA 和 C 端 11-AA 截断的 AgsA 混合时（各自有效伴侣活性所需量的一半），所有底物和温度下都观察到良好的伴侣活性。使用荧光共振能量转移分析在 25°C 时未检测到 AgsA 寡聚体之间的可检测分子间交换；然而，在 25°C 时观察到 AgsA 寡聚体与 C 端截断的 AgsA 之间的显著交换。
我们的数据表明，AgsA 具有几个对有效伴侣活性至关重要的区域：对溶菌酶伴侣活性重要的区域位于寡聚复合物的外表面，而对胰岛素重要的区域位于寡聚复合物的内部，对 MDH 重要的区域位于 N 端臂内。此外，寡聚体和二聚体结构之间的平衡似乎对其有效的伴侣活性很重要。
