Department of Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
Molecules. 2023 Jun 9;28(12):4663. doi: 10.3390/molecules28124663.
Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.
目前，人们对新的多亚基笼状蛋白和球形病毒衣壳的发现和应用产生了浓厚的兴趣，将其应用于生物纳米技术、药物输送和诊断成像领域，因为它们的内部腔室可以作为荧光团或生物活性分子货物的宿主。在铁储存笼状蛋白的铁蛋白蛋白超家族中，细菌铁蛋白是不寻常的，因为它含有 12 个血红素辅因子并且是同型的。本研究的目的是通过开发利用细菌铁蛋白进行分子货物包封的新方法来扩展铁蛋白的功能。探索了两种策略来控制与随机包封相比，广泛的分子客体的包封，随机包封是该领域中采用的主要策略。第一种策略是在细菌铁蛋白的内部腔室内包含组氨酸标记肽融合序列。这种方法允许成功且受控地包封荧光染料、蛋白质（荧光标记的链霉亲和素）或 5nm 金纳米颗粒。第二种策略称为血红素依赖性盒策略，涉及用与（i）荧光染料或（ii）镍-亚氨基二乙酸（NTA）基团连接的血红素类似物替代天然血红素（这允许可控制地包封带有组氨酸标记的绿色荧光蛋白）。一种基于计算机对接的方法确定了几种能够替代血红素并能够控制蛋白质四级结构的小分子。还完成了基于转谷氨酰胺酶的化学酶表面修饰这种笼状蛋白的方法，允许未来的纳米颗粒靶向。这项研究提出了控制各种分子包封的新策略，并为内部蛋白腔工程增加了另一个复杂程度。
