Understanding, engineering and exploiting protein self-assembly.

了解、设计和利用蛋白质自组装。

• 批准号: RGPIN-2017-05907
• 负责人: Rainey, Jan
• 金额: $ 3.64万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662912
• 关键词: Understanding; engineering; exploiting; protein; self

This NSERC-funded research program focuses on developing an atomic-level understanding of how spider silks, a type of protein, are able to form larger structures such as fibres and particles. Beyond expanding understanding of these poorly understood formation processes, the long-term goal of these studies is the engineering of proteins specifically tailored to a desired form. This provides an outstanding potential range of materials for biotechnological use.******Spider aciniform silk is a protein fibre used by spiders to wrap their prey. It is one of the toughest known materials, meaning that it can absorb more energy prior to breaking than almost any other material. The more widely-studied spider dragline silk, in contrast, can withstand heavier loads but is not as flexible, giving a lower toughness.******Harvesting of silk from spiders is not a feasible route to obtain sufficient amounts of material for applied uses. We have developed a method to use bacteria to produce this silk. This has allows us to readily modify the individual amino acids making up the protein and to label the protein to allow modern nuclear magnetic resonance (NMR) spectroscopy experiments. To date, we have bacterially produced a variety of recombinant aciniform silk proteins, developed methods to produce fibres with a diameter ~10% that of human hair and >1 km in length, and have formed tiny silk particles ("nanoparticles") that can be loaded with different chemicals. These silk fibres have significant potential for application in a wide variety of areas given their outstanding toughness. Alternatively, our silk nanoparticles are potentially applicable for drug delivery or crop protection applications.******For each form of silk protein, we use NMR spectroscopy to pinpoint the locations of individual atoms. This has allowed us to determine the atomic-level structure of aciniform silk in solution. We can also monitor specific changes to atomic positions and motion during the transition from the soluble state to the fibrous state. As we do so, we monitor the types and properties of fibres or nanoparticles that are formed using atomic force and electron microscopy. We also apply tensile testing methods to determine strength and toughness. Comparison of the atomic arrangement to mechanical properties as a function of changes in amino acids allows us to engineer new forms of silk based upon acinform silk but incorporating specific, desirable properties.******Beyond these scientific and materials outcomes, this DG-funded work allows highly qualified personnel from undergraduate through postdoctoral researchers to become expert in techniques to: (1) bacterially produce proteins; (2) characterize the atomic-level behaviour of proteins in soluble states, nanoparticles, and silk fibres; (3) spin silk proteins into fibres; (4) characterize fibre and particle appearance and mechanics; and, (5) engineer proteins with desired chemical properties.

这个由NSERC资助的研究项目专注于开发一种原子水平的理解，即蜘蛛丝（一种蛋白质）如何能够形成更大的结构，如纤维和颗粒。除了扩大对这些知之甚少的形成过程的理解之外，这些研究的长期目标是专门针对所需形式设计蛋白质。这为生物技术应用提供了一个突出的潜在材料范围。**蜘蛛腺泡状丝是蜘蛛用来包裹猎物的蛋白质纤维。它是已知最坚硬的材料之一，这意味着它可以在断裂之前吸收比几乎任何其他材料更多的能量。相比之下，更广泛研究的蜘蛛拖丝可以承受更重的负荷，但没有那么灵活，韧性较低。从蜘蛛身上收获丝并不是获得足够数量的材料用于应用的可行途径。我们已经开发出一种利用细菌生产这种丝的方法。这使我们能够很容易地修改组成蛋白质的单个氨基酸，并标记蛋白质以允许现代核磁共振（NMR）光谱实验。到目前为止，我们已经通过细菌生产了各种重组腺泡状丝蛋白，开发了生产直径约为人类头发直径10%且长度>1公里的纤维的方法，并形成了可以装载不同化学物质的微小丝颗粒（“纳米颗粒”）。这些蚕丝纤维具有显著的应用潜力，因为它们具有出色的韧性。或者，我们的丝纳米颗粒可能适用于药物输送或作物保护应用。对于每种形式的丝蛋白，我们使用NMR光谱来确定单个原子的位置。这使我们能够确定溶液中腺泡状丝的原子级结构。我们还可以监测从可溶态到纤维态转变期间原子位置和运动的具体变化。当我们这样做时，我们监测使用原子力和电子显微镜形成的纤维或纳米颗粒的类型和性质。我们还应用拉伸测试方法来确定强度和韧性。将原子排列与机械性能作为氨基酸变化的函数进行比较，使我们能够在acinform丝的基础上设计新形式的丝，但结合特定的，理想的性能。除了这些科学和材料成果之外，DG资助的这项工作还使高素质的本科生到博士后研究人员成为技术专家：（1）细菌生产蛋白质;（2）表征可溶态蛋白质，纳米颗粒和丝纤维的原子水平行为;（3）将丝蛋白纺成纤维;（4）表征纤维和颗粒的外观和力学;（5）表征纤维和颗粒的外观和力学。以及（5）将蛋白质改造成具有所需化学性质。