Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Department of Biological Engineering, Inha University, 100 Inha-ro, Michuhol-gu, Incheon 22212, Republic of Korea.
Biomacromolecules. 2021 Feb 8;22(2):289-298. doi: 10.1021/acs.biomac.0c01083. Epub 2021 Jan 11.
Natural selective filtering systems (e.g., the extracellular matrix, nuclear pores, and mucus) separate molecules selectively and efficiently, and the detailed understanding of transport mechanisms exploited in these systems provides important bioinspired design principles for selective filters. In particular, nucleoporins consist of consensus repeat sequences that are readily utilized for engineering repeat proteins. Here, the consensus repeat sequence of Nsp1, a yeast nucleoporin, is polymerized to form a nucleoporin-like protein (NLP) and mutated to understand the effect of sequence on selective transport. The hydrophilic spacers of the NLPs were redesigned considering net charge, charge distribution, and polarity. Mutations were made near to and far from the FSFG interacting domain to explore the role of highly conserved residues as a function of spatial proximity. A nuclear transport receptor-cargo complex, nuclear transport factor 2-green fluorescent protein (NTF2-GFP), was used as a model for changes in transport. For mutations of the charged spacer, some mutations of highly conserved charged residues were possible without knocking out selective transport of the NTF2, but the formation of regions of clustered negative charge has an unfavorable effect on nuclear transporter permeation. Thus, positive net charge and alternating positive and negative charge within the hydrophilic spacer are advantageous for recognition and selective transport. In the polarity panel, mutations that increased the interaction between NTF2-GFP and the gel led to decreased permeation of the NTF2-GFP due to blocking of the interface and inability of the NTF2-GFP to transport into the gel. Therefore, these results provide a strategy for tuning selective permeability of biomolecules using the artificially designed consensus repeat-based hydrogels.
天然选择性过滤系统（例如细胞外基质、核孔和黏液）能够选择性和有效地分离分子，而对这些系统中所利用的传输机制的详细了解为选择性过滤器提供了重要的仿生设计原则。特别是核孔蛋白由共识重复序列组成，这些序列很容易被用于工程化重复蛋白。在这里，酵母核孔蛋白 Nsp1 的共识重复序列被聚合形成核孔蛋白样蛋白（NLP），并进行突变以了解序列对选择性传输的影响。考虑到净电荷、电荷分布和极性，重新设计了 NLPs 的亲水性间隔区。在靠近和远离 FSFG 相互作用域的位置进行突变，以探索高度保守残基的作用与空间接近度的关系。核转运受体-货物复合物，核转运因子 2-绿色荧光蛋白（NTF2-GFP）被用作转运变化的模型。对于带电荷间隔区的突变，一些高度保守的带电荷残基的突变可以在不破坏 NTF2 选择性运输的情况下进行，但形成聚集的负电荷区域对核转运体渗透有不利影响。因此，亲水性间隔区内的净正电荷和正负电荷交替出现有利于识别和选择性运输。在极性面板中，增加 NTF2-GFP 与凝胶之间相互作用的突变会导致 NTF2-GFP 的渗透率降低，这是由于界面被阻塞以及 NTF2-GFP 无法进入凝胶导致的。因此，这些结果为使用人工设计的基于共识重复的水凝胶来调整生物分子的选择性渗透性提供了一种策略。
