Rational Design of Complex Protein Functions by Modulation of Backbone Dynamics

蛋白质在研究，工业和医学中有着广泛的应用，因为它们能够以极高的精度和效率介导复杂的分子过程。虽然人们通常认为蛋白质的功能是由其三维结构决定的，但越来越多的证据表明，溶液中随时间变化的结构波动（即动力学）对蛋白质的复杂功能至关重要。目前，合理设计这些复杂的功能以满足工业和医学不断增长的需求是极具挑战性的，因为我们缺乏预测蛋白质序列的方法，这些蛋白质序列将显示必要的动力学。已经开发了计算蛋白质设计（CPD）技术来鉴定可以采用特定三维结构并具有所需性质（例如增加的稳定性、改善的结合亲和力或改变的特异性）的序列。然而，标准的CPD方法评估单一固定蛋白质结构上的序列，这不允许明确考虑动力学，从而防止按需合理设计复杂蛋白质功能。在这个拟议的研究计划中，我们将率先开发用于调节蛋白质动力学的多态设计（MSD）方法，提高可以通过理性设计访问的功能的复杂性。MSD是CPD中的一种新兴方法，它考虑任何数量的蛋白质结构作为计算的输入，而不是单个蛋白质骨架模板。例如，近似蛋白质柔性的骨架集合可以用作MSD中的输入结构，为需要考虑动力学的复杂蛋白质功能的设计打开了大门。在NSERC的资助下，我们将开发一个MSD框架来随意调节任何蛋白质的动力学，并将使用它来设计G1测试蛋白的突变体，通过硬化其桶结构来显示增加或减少的动力学和更亮的红色荧光蛋白。这些研究项目将由一名博士后研究员、三名研究生和十名本科生组成的团队进行。我们的成功将构成蛋白质工程的突破性发展，为将结构可塑性整合到广泛的设计功能（如构象转换和酶催化）中打开大门。我们的工作还将促进我们对蛋白质结构，动力学和功能之间联系的理解，从而产生对整个蛋白质科学界产生深远影响的基本见解。从长远来看，我们的工作将使我们更接近“按需设计蛋白质”的时代，产生应对21世纪加拿大在工业，环境，农业和医学方面的重要挑战所需的新技术。

Proteins have found widespread application in research, industry, and medicine because of their ability to mediate complex molecular processes with extreme precision and efficiency. While it is generally assumed that the function of a protein is dictated by its three-dimensional structure, increasing evidence demonstrates that time-dependent structural fluctuations in solution (i.e. dynamics) are essential to complex functions of proteins. Currently, it is extremely challenging to rationally design these complex functions to meet ever-growing needs in industry and medicine because we lack methods to predict protein sequences that will display the necessary dynamics. Computational protein design (CPD) techniques have been developed to identify sequences that can adopt a specific three-dimensional structure and possess a desired property such as increased stability, improved binding affinity, or altered specificity. However, standard CPD methods evaluate sequences on a single fixed protein structure, which does not allow for the explicit consideration of dynamics, thereby preventing the rational design of complex protein functions on demand. In this proposed research program, we will pioneer the development of multistate design (MSD) methods for the modulation of protein dynamics, enhancing the complexity of functions that can be accessed by rational design.***MSD is an emerging methodology in CPD that considers any number of protein structures as inputs to calculation instead of a single protein backbone template. For example, backbone ensembles approximating protein flexibility can be used as input structures in MSD, opening the door to the design of complex protein functions that require the consideration of dynamics. With funding from NSERC, we will develop a MSD framework to modulate dynamics of any protein at will, and will use it to design mutants of the G1 test protein displaying increased or decreased dynamics and brighter red fluorescent proteins by rigidifying their barrel structure. These research projects will be carried out by a team composed of a postdoctoral fellow, three graduate students, and ten undergraduates. Our success will constitute a ground-breaking development in protein engineering, opening the door to the integration of structural plasticity into a wide range of designed functions such as conformational switching and enzymatic catalysis. Our work will also advance our understanding of the link between protein structure, dynamics, and function, leading to fundamental insights that will have far-reaching impact in the protein science community at large. In the longer term, our work will take us one step closer to an age of designer proteins on-demand', yielding new technologies required to tackle important challenges of 21st century Canada in industry, environment, agriculture, and medicine.**