Rational Design of Complex Protein Functions by Modulation of Backbone Dynamics

蛋白质在研究、工业和医学中得到了广泛的应用，因为它们能够以极高的精度和效率调节复杂的分子过程。虽然一般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日益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态。计算蛋白设计（CPD）技术已经发展到可以识别序列，可以采用特定的三维结构，并具有期望的性质，如增加的稳定性，改善的结合亲和力，或改变特异性。然而，标准的CPD方法评估单个固定蛋白质结构上的序列，不允许明确考虑动力学，从而阻碍了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蛋白质动力学调节的多状态设计（MSD）方法，提高可以通过合理设计访问的功能的复杂性。

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 Gß1 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.