Humenik Martin, Magdeburg Michael, Scheibel Thomas
Lehrstuhl Biomaterialien, Fakultät für Ingenieurwissenschaften, Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany.
Lehrstuhl Biomaterialien, Fakultät für Ingenieurwissenschaften, Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany.
J Struct Biol. 2014 Jun;186(3):431-7. doi: 10.1016/j.jsb.2014.03.010. Epub 2014 Mar 20.
Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.
重组蜘蛛丝变体eADF4(Cn)由天然园蛛的拖牵丝ADF4衍生而来，包含不同数量（n）的共有序列基序C。当n⩾2时，所产生的纳米纤维无法区分。C模块包含35个富含甘氨酸和脯氨酸残基的氨基酸（以GPGXY重复序列形式存在）以及一个（Ala）8聚丙氨酸片段。所有变体在溶液中均表现为内在无序状态，在形成纤维时会转变为交叉β结构。异源接种表明不同的eADF4(Cn)变体之间具有高度的结构兼容性，然而，它们的组装动力学因重复次数而异。动力学分析揭示了一种典型的交叉β纤维形成的成核-生长机制，成核速率和生长速率均随着重复次数的增加而增加。引人注目的是，单个C模块不会自组装成纤维，但添加异源种子后可观察到纤维生长。显然，至少两个C模块的相互连接显著促进了从无序状态到β折叠结构的结构转变，这对于成核是必要的，并且有利于纤维生长。
