Moradian-Oldak J, Paine M L, Lei Y P, Fincham A G, Snead M L
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, California, Los Angeles 90033, USA.
J Struct Biol. 2000 Jul;131(1):27-37. doi: 10.1006/jsbi.2000.4237.
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 amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-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.
动态光散射（DLS）分析与原子力显微镜（AFM）成像相结合，用于研究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。分析了缺少据信参与自组装的氨基或羧基末端结构域的小鼠釉原蛋白，以及具有与牙釉质发育不全病例中发现的相同的单氨基酸或双氨基酸突变的釉原蛋白。含多组氨酸的全长重组釉原蛋白 [rp(H)M180] 产生了具有单分散尺寸分布的纳米球（根据DLS估计的流体动力学半径为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形成的纳米球相当，表明这种组氨酸修饰不干扰自组装过程。从釉原蛋白中删除N末端自组装结构域并用FLAG表位替代（“A”结构域缺失）导致形成具有异质尺寸分布的聚集体，颗粒的流体动力学半径范围为3至38 nm。对缺少氨基酸157至173并含有血凝素表位的釉原蛋白分子（“B”结构域缺失）进行时间依赖性动态光散射分析，结果形成了颗粒（21.5±6.8 nm），这些颗粒在一小时内融合形成更大的49.3±4.3 nm的颗粒。N末端区域的单点和双点突变导致形成更大且更不均匀的纳米球。上述数据表明，虽然N末端A结构域参与纳米球形成的分子相互作用，但羧基末端B结构域有助于纳米球的稳定性和均匀性，防止它们融合成更大的聚集体。这些体外研究结果支持这样的观点，即在牙釉质形成过程中发生的釉原蛋白在氨基和羧基末端的蛋白水解切割会影响自组装过程中釉原蛋白与釉原蛋白之间的相互作用，从而改变发育中的牙釉质细胞外基质的结构组织，进而影响牙釉质生物矿化。
