Biomimetic Crystallisation of Metal-Organic Materials for Protein Isolation and Stabilization

蛋白质已经进化了数百年，执行各种各样的高度复杂的任务，但它们的结构脆弱性继续限制它们在生物环境之外的应用。因此，如果要实现利用蛋白质的全部潜力，则开发能够支持蛋白质的三维结构的封装策略是必不可少的，所述蛋白质的三维结构通常会产生其期望的性质。具体而言，蛋白质稳定化带来的益处可预见于：i）在制造业中，酶可能能够取代目前使用的昂贵且对环境有毒的催化剂; ii）在医疗保健中，纯化和不稳定性问题提高了蛋白质药物的成本，限制了其在长期疾病管理中的应用及其在第三世界的使用;以及iii）在研究科学中，生物学家努力了解酶如何在有限空间内发挥作用。该提案将系统地评估最适合在天然状态下包封和稳定蛋白质的材料特性。提出了一种生物启发的结晶方法，其中金属有机框架（MOFs）在溶液中围绕蛋白质组装，从而避免了与具有足够大的内部空隙袋以容纳蛋白质的MOFs的合成和稳定性相关的限制。在项目的初始阶段将筛选各种条件，以评估最适合MOFs仿生结晶的金属，有机配体，结晶条件和蛋白质性质，而不会对蛋白质产生负面影响。将评价纯化蛋白质以及引入复杂生物混合物中的蛋白质的包封。优化结晶条件后，将包封一系列酶，并评价包封酶与未包封酶的稳定性和酶促反应速率的变化。假设包封将增加酶的稳定性，但由于与分子通过框架材料的运动相关的限制，可能降低或改变催化反应的速率和反应范围。最后，将对稳定框架材料的受限空间内的蛋白质进行详细表征，并评估表征通常显示高度紊乱的生物蛋白质或已知在受限空间内工作的生物蛋白质的潜在机会。

Proteins have evolved over hundreds of years to perform a wide variety of highly sophisticated tasks, however their structural fragility continues to limit their application beyond biological settings. Development of an encapsulation strategy capable of supporting the three-dimensional structure of proteins that often gives rise to their desirable properties, is therefore essential if the full potential to utilise proteins is to be realised. Specifically, benefits arising from protein stabilisation are foreseen in:i) the manufacturing sector where enzymes may be able to replace expensive and environmentally toxic catalysts currently in use, ii) in healthcare where purification and instability issues raise the cost of protein pharmaceuticals limiting their application for long-term disease management and their use in the Third World, and also iii) in research science where biologists strive to understand how enzymes function within confined spaces. This proposal will systematically evaluate the materials properties most suited for encapsulating and stabilising proteins in their native state. A biologically inspired crystallisation methodology is proposed where metal-organic frameworks (MOFs) assemble around proteins in solution, thus avoiding limitations associated with the synthesis and stability of MOFs with sufficiently large internal void pockets to accommodate proteins. A wide variety of conditions will be screened in the initial stages of the project to evaluate the metal, organic ligand, crystallisation conditions and protein properties best suited to biomimetic crystallisation of MOFs without negatively impacting the protein. Encapsulation of purified proteins as well as those introduced in complex biological mixtures will be evaluated.Following optimisation of the crystallisation conditions a range of enzymes will be encapsulated and changes in the stability and rate of enzymatic reaction for the encapsulated enzyme versus the non-encapsulated enzyme will be evaluated. It is hypothesised that encapsulation will increase the stability of the enzyme, but may reduce or modify the rate of the catalysed reaction and the reaction scope due to limitations associated with the movement of molecules through the framework material. Finally, detailed characterisation of the protein within the restricted space of the stabilising framework material will be undertaken and potential opportunities for characterisation of biological proteins that typically display a high degree of disorder or those that are known to operate within a confined space will be evaluated.