Molecular Mechanisms and Design of Protein Folding, Function, and Aggregation

蛋白质折叠、功能和聚集的分子机制和设计

• 批准号: RGPIN-2022-05139
• 负责人: Meiering, Elizabeth
• 金额: $ 6.7万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748012
• 关键词: Molecular; Mechanisms; Design; Protein; Folding

Proteins are exquisite molecular machines - they perform a vast range of functions, from acting as catalysts, to specifically recognizing and binding all kinds of molecules, to structural support roles. The primary amino acid sequence of a protein determines how it folds to its 3D structure, which in turn determines its function.  Despite extensive study, the details of the molecular mechanisms governing protein folding, aggregation and function remain obscure. Defining these mechanisms is of tremendous importance because such knowledge will ultimately enable the precise prediction and control of the specific properties of any protein: in natural processes, in disease and in biotechnology. It will give us the ability to engineer or design proteins for any desired application! Natural and engineered proteins are widely used in research and industry and are transforming biotechnology through their applications in material science, and as catalysts and drugs. Explosive growth in knowledge of protein sequences, structures and functions and in computational modeling of proteins has set the stage for uncovering the molecular mechanisms that determine protein properties. For example, based on our experimental measurements of protein solubility, we recently developed new structure-based methods to predict and design the solubility of engineered target binding proteins known as Adnectins. As for many proteins, Adnectins form insoluble inclusion bodies (IBs) when expressed in bacteria, and the level of IB formation is related to the solubility of the pure protein. Our findings for Adnectins are important for controlling solubility when developing Adnectins for medical applications. In addition, we advanced a method for high resolution structure analysis of IBs using quenched H/D exchange nuclear magnetic resonance spectroscopy (NMR). Applying this method revealed native-like structure in IBs, which has important implications for developing IBs as functional materials. In a separate project, we developed a combined NMR and molecular dynamics computational method that revealed at atomic resolution the mechanism of pH-dependent switching function in Hisactophilin, which controls cell movement. This powerful method is widely applicable to defining local structural stability and the population of alternative states that control protein structure and function. Our discoveries for these and other proteins (including numerous mutants which we design to systematically grow our knowledge) are obtained using a battery of experimental (spectroscopy, calorimetry, light scattering) and computational tools.  We will build on our previous exciting discoveries to advance knowledge of how proteins work and how they can be harnessed for a myriad of applications; this research will provide valuable collaborative interdisciplinary and international training for many highly qualified personnel in developing proteins for further great benefit to society.

蛋白质是精致的分子机器-它们执行广泛的功能，从充当催化剂，到特异性识别和结合各种分子，再到结构支持角色。蛋白质的一级氨基酸序列决定了它如何折叠成三维结构，而三维结构又决定了它的功能。尽管有广泛的研究，但关于蛋白质折叠、聚集和功能的分子机制的细节仍然不清楚。定义这些机制是非常重要的，因为这些知识将最终能够精确预测和控制任何蛋白质的特定特性：在自然过程中，在疾病和生物技术中。它将使我们有能力为任何所需的应用工程或设计蛋白质！天然和工程蛋白质广泛用于研究和工业，并通过其在材料科学中的应用以及作为催化剂和药物而改变生物技术。蛋白质序列、结构和功能知识的爆炸性增长以及蛋白质的计算建模为揭示决定蛋白质性质的分子机制奠定了基础。例如，基于我们对蛋白质溶解度的实验测量，我们最近开发了新的基于结构的方法来预测和设计被称为Adnectin的工程化靶结合蛋白的溶解度。对于许多蛋白质，Adnectin在细菌中表达时形成不溶性包涵体（IB），并且IB形成的水平与纯蛋白质的溶解度有关。我们对Adnectin的发现对于在开发Adnectin用于医疗应用时控制溶解度非常重要。此外，我们还提出了一种利用淬灭H/D交换核磁共振谱（NMR）进行高分辨率结构分析的方法。应用该方法揭示了IBs的类天然结构，这对IBs作为功能材料的开发具有重要意义。在一个单独的项目中，我们开发了一种结合NMR和分子动力学计算方法，以原子分辨率揭示了Hisactophilin中pH依赖性开关功能的机制，该机制控制细胞运动。这种强大的方法广泛适用于定义局部结构稳定性和控制蛋白质结构和功能的替代状态的群体。 我们对这些和其他蛋白质的发现（包括我们设计的许多突变体，以系统地增长我们的知识）是使用一系列实验获得的。（光谱学，量热法，光散射）和计算工具。我们将建立在我们以前令人兴奋的发现，以推进蛋白质如何工作以及如何利用它们进行无数应用的知识;这项研究将为许多高素质的蛋白质开发人员提供宝贵的跨学科和国际合作培训，为社会带来更大的利益。