Exploring the Impact of Backbone Flexibility on Folding Mechanisms of Protein Mimetics: Integrating Experiment and Simulation

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With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Dr. Seth Horne and Dr. Lillian Chong from the University of Pittsburgh to characterize folding mechanisms in artificial, protein-like molecules. Proteins, the intricate molecular machines that perform the majority of functions necessary to sustain life, are made up of combinations of molecular building blocks known as amino acids, arrayed in long chains. Nature provides twenty different amino acid molecules, and variations in the sequence in which they are chained together leads proteins to adopt diverse three-dimensional folded shapes. These folded shapes, in turn, dictate protein function. In recent years, researchers have shown that a variety of artificial protein backbones, where some of the natural amino acids are replaced by different molecules of approximately the same size, can fold to adopt complex protein-like shapes. Artificial backbones have fundamental value in helping understand how proteins work and also offer practical benefits over their natural counterparts. Small proteins currently find widespread use as agents for the diagnosis and treatment of disease; however, an important limitation to such application is rapid degradation by enzymes that are abundant in the bloodstream. Changing the backbone while keeping side chains intact can create a molecule that mimics the folded shape and biological function of a prototype natural protein but is less prone to degradation in the body. A crucial component in the design of artificial backbones with predictable shapes is understanding how they fold. This project seeks to elucidate the molecular mechanism by which artificial backbones fold through closely-coupled experiments and computational modeling. This work includes the development of a new force field---the mathematical model describing interactions among the natural and artificial amino acids---for the modeling component of the research. The force field will be disseminated as open source through the widely-used AMBER molecular dynamics simulation package. This interdisciplinary, collaborative project is providing a valuable training ground for graduate and undergraduate students participating in the research, and is supporting diverse educational and outreach activities, including a summer undergraduate creative science writing workshop, guest lectures on issues related to scientific research and science communication, and a three-week workshop on Python coding at a local high school. The creative science writing workshop is being designed and offered by the Chemistry, English, and the History and Philosophy of Science Departments at the University of Pittsburgh, and provides students with joint science and writing mentorship, culminating in submission of one or more capstone pieces for publication in news outlets with broad readership.A key recent advance that pushed the frontier of structural complexity possible in protein mimetics is the finding that backbone connectivity can be substantially altered without compromising the fold specified by a natural side-chain sequence. Compared to the growing body of structural information on such "heterogeneous-backbone" protein mimics, virtually nothing is known about how backbone alteration impacts dynamics or folding pathways. Addressing this gap in knowledge has the potential to reveal new insights into the folding behavior in artificial backbones, aid in the design of more effective protein mimics, and provide insights into fundamental issues related to natural protein folding. This project is determining how protein backbone connectivity influences chain dynamics and folding mechanisms. The research is guided by the central hypothesis that a combination of experimental biophysical analysis and atomistic computer simulations can reveal the complex interplay among backbone composition, folded structure, chain dynamics, solvation, and folding pathways. The project includes the design and parameterization of a modified AMBER force field for simulating folding and molecular recognition events involving artificial protein-like backbones, and the development of novel protocols for simulating protein folding processes using the weighted ensemble enhanced sampling strategy. The force field is being disseminated as part of the AMBER molecular dynamics package. The new simulation strategies are being used to model the thermodynamic, structural, and kinetics experiments performed on corresponding protein mimetics synthesized in the PI's lab. This unique combination of experimental and computational research is providing insights into the molecular origins of stability differences between natural proteins and artificial counterparts, folding behavior in artificial-backbone protein mimetics, and the fundamental role of backbone preorganization in protein folding. The project is providing research training opportunities for high school, undergraduate, and graduate students in state-of-the-art protein design and modeling. An undergraduate Creative Science Summer Program at the University of Pittsburgh is being expanded, enabling students to develop valuable writing skills in the sciences, to communicate the critical roles that science plays in society.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.