Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations
Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations
复制标题
DOI：
10.1159/000324342
复制
发表时间：
2011-01-01
影响因子：
2.7
通讯作者：
Moradian-Oldak, Janet
中科院分区：
文献类型：
--
作者：
Bromley, Keith M.;Lakshminarayanan, Rajamani;Moradian-Oldak, Janet
在 2 例牙釉质生成不全病例中，从人类 DNA 序列中鉴定出了牙釉蛋白内的两个点突变（T21I 和 P40T）。我们研究了重组牙釉蛋白 (rM180) 的折叠和自组装，并与 T21I 和 P40T 突变体类似物进行比较。在pH 5.8和25℃下，rM180和P41T突变体作为单体存在，而T21I突变体形成小寡聚体。在 pH 8 和 25 摄氏度下，所有牙釉蛋白样品均形成流体动力学半径 (R(H)) 约为 15-16 nm 的纳米球。加热至 37 摄氏度后，P41T 颗粒尺寸增大（R(H) = 18 nm）。在 pH 5.8 的热变性过程中，两种突变蛋白的重折叠速度都比野生型 (WT) rM180 慢。变温色氨酸荧光和动态光散射研究表明，WT 在加热后转变为部分折叠构象并保持稳定。热变性和重折叠研究表明，与 WT 相比，突变体稳定性较差，并且表现出更强的过早聚集能力。我们的数据表明，就 P41T 而言，牙釉蛋白自组装的改变是二级结构不稳定的结果，而就 T21I 而言，则是 N 端区域整体疏水性变化的结果。我们认为，突变型牙釉蛋白组装（即过早聚集）的改变可能对细胞内和细胞外过程产生深远的影响，例如牙釉蛋白分泌、蛋白水解及其与非牙釉蛋白以及形成矿物质的相互作用。版权所有 (C) 2011 S. Karger AG，巴塞尔
Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25 degrees C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25 degrees C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R(H)) of around 15-16 nm. Upon heating to 37 degrees C, particles of P41T increased in size (R(H) = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wildtype (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra-and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral. Copyright (C) 2011 S. Karger AG, Basel