Engineered biological systems, ranging from molecules with new functions to entire organisms, have tremendous practical importance; they can also fundamentally change how we ask questions about the biological principles of function. Our research aims to invent approaches to engineer new molecules that operate as predicted in biological contexts, and to utilize prediction and engineering to address fundamental questions on the relationship of molecular mechanisms and systems-level function. To address the many current challenges in the field – from developing more predictive computational design methods to determining the requirements for function in cells – we combine concepts from computer science, physics, chemistry, mathematics, engineering and biology.
Our work spans three interrelated themes:
I. Develop computational methods for modeling & design of proteins, in the program Rosetta (www.rosettacommons.org).
Predicting and designing the structures of proteins with biologically useful accuracy has been a key challenge in computational structural biology and molecular engineering. We have made methodological advances that address one of the main bottlenecks: sampling the vast number of conformations accessible to proteins. We have utilized a method for moving through conformational space inspired by principles from robotics – a field with a rich history in efficient calculation of mechanically accessible states subject to constraints. We applied the same mathematics that can be used to direct the motions of a robot arm to compute the degrees of freedom of a polypeptide chain (Mandell et al., Nature Methods 2009). Our predictions generate hypotheses on protein conformations controlling biological processes – such as protein recognition, signal transduction, and enzyme active site gating – and are laying the foundation for our work reengineering and “reshaping” protein interfaces and active sites for new functions. From March-August 2020, we rapidly deployed our methods to engineer ACE2-based receptor traps that potently neutralize live SARS-CoV-2 virus in cell culture as therapeutic candidates for further development (Glasgow et al., PNAS 2020). Most recently, we developed an approach to engineer a vast universe of new proteins with tunable shapes (Pan et al., Science 2020).
II. Create new proteins and devices with more advanced functions by experimental engineering.
Designer molecules with new biological functions could have many exciting uses: protein therapeutics with minimal side effects; new enzymes and biological synthesis pathways for fuel molecules or compounds that are otherwise too expensive to produce; sensor/actuator devices that can report on cell biological processes in real time; robust signaling systems that can detect specific inputs and generate a precise response; protein machines that can be controlled by specific external inputs such as light. Over the past several years, we have engineered a range of proteins with new functions, including protein-protein interactions that are specific enough to control complex biological processes in mammalian cells (Kapp, Liu et al., PNAS, 2012). We have also engineered proteins whose functions can be switched by phosphorylation or light. One example is a study describing the control of precise shape transitions of a large protein assembly with optical inputs, where we successfully exchanged the ‘engine’ of a protein-based ATP-driven molecular machine to be powered by light (Hoersch et al., Nature Nanotechnology 2013). Most recently, we have focused on application of computational protein design to endow cells with the ability to sense and respond to new molecular signals and orchestrate desired biological responses, one of the most fundamental capabilities of living systems (Glasgow, Huang, Mandell et al., Science 2019).
III. Dissect design principles of function in cells by combining prediction and engineering approaches.
The complexity of biological interaction networks makes it impossible at present to accurately predict the effects of perturbations to the network, such as expression changes or mutations, and, consequently, how to intervene effectively when networks are misregulated. To address this problem, we have become increasingly interested in approaches that quantify the complex cellular consequences of perturbations to biological networks together with their underlying mechanisms. In 2012, we published a study on engineered perturbations to a classic system for regulatory mechanisms of gene expression: the lac operon (Eames & Kortemme, Science 2012). A current experimental effort is directed towards determining the system-level functions of specific interactions in cells and organisms by systematically modulating protein interactions and protein abundance. In collaboration with Nevan Krogan’s laboratory at UCSF, we have made systematic mutational perturbations in a central GTPase molecular switch conserved in all eukaryotes (Perica, Mathy et al., bioRxiv 2021). By integrating functional genetic and proteomics approaches with biophysics, we find that distinct biological processes are differentially sensitive to the kinetics of switching, addressing the long-standing question of how a single molecular switch can differentially regulate different cellular functions. We hope that these principles of cellular control can guide cellular engineering, and ultimately lead to new and more precise ways to counteract misregulation in disease.