Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy
Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy
复制标题
DOI：
10.1006/jsbi.2000.4237
复制
发表时间：
2000-07-01
影响因子：
3
通讯作者：
Snead, ML
中科院分区：
文献类型：
--
作者：
Moradian-Oldak, J;Paine, ML;Snead, ML
采用动态光散射(DLS)和原子力显微镜(AFM)相结合的方法研究了一系列重组釉原蛋白的超分子自组装特性。总体目标是确定釉原蛋白中的某些结构基序在自组装过程中对蛋白质-蛋白质相互作用的贡献。分析了缺乏氨基或羧基末端结构域的小鼠釉原蛋白，以及具有与自闭症不全病例相同的单氨基酸或双氨基酸突变的釉原蛋白，含有多组氨酸的全长重组釉原蛋白[RP(H)M180]产生了尺寸分布单一的纳米球(根据DLS估计流体动力学半径为20.7+/-2.9 nm，根据AFM图像估计为16.1+/-3.4 nm)，与不含多组氨酸结构域的全长釉原蛋白rM179形成的纳米球相似，表明这种组氨酸修饰不会干扰自组装过程。从釉原蛋白中删除N-末端自组装结构域并用标志替换它们；表位(‘’A‘-结构域缺失)导致形成具有不均匀尺寸分布的集合体，其颗粒的流体动力学半径范围为3-38 nm。对缺少157到173个氨基酸的含有血凝素表位的釉原蛋白分子进行随时间变化的动态光散射分析发现，在1小时内形成的颗粒(21.5+/-6.8 nm)融合成49.3+/-4.3 nm的较大颗粒。N-末端区域的单点和双点突变导致了更大更多的纳米球的形成，上述数据表明，虽然N-末端-A结构域参与了纳米球形成的分子相互作用，但羧基末端的B-结构域有助于纳米球的稳定性和均一性，阻止了它们与更大的集合体融合。这些体外研究结果支持这一观点，即在釉质形成过程中，釉原蛋白在氨基和羧基末端的蛋白水解性切割会影响自组装过程中釉原蛋白与釉原蛋白的相互作用，从而改变发育中的釉质细胞外基质的结构组织，从而影响釉质的生物矿化。(C)2000年学术出版社。
Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of antelogenesis imperfecta were analyzed, The polyhistidine-containing full-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG; epitope (''A'"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres, The above data suggest that while the N-terminal-A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization. (C) 2000 Academic Press.