Rational Design of Complex Protein Functions by Modulation of Backbone Dynamics

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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.**