Designing proteins that can change their structure, and ultimately function, in response to specific molecular signals, a phenomenon known as allosteric regulation, has been a long-standing goal of protein engineering.
In a new paper published in Nature, entitled “De novo design of allosterically switchable protein assemblies,” researchers from the University of Washington led by renowned structural biologist, David Baker, PhD, professor of biochemistry and the director of the Institute for Protein Design at the University of Washington (UW) Medicine, have engineered proteins that can reliably and accurately transition between assembly and disassembly through allosteric control. Leveraging AI to design new proteins not found in nature, the authors engineered a wide variety of dynamic protein arrangements to provide a roadmap for applications in triggerable delivery systems, biosensing, cellular feedback control circuitry, and more.
“By designing proteins that can assemble and disassemble on command, we pave the way for future biotechnologies that may rival even nature’s sophistication,” said Baker.
Historically, engineering proteins with allosteric regulation relied on coupling nature’s existing proteins, which limited the breadth of accessible protein functions. In contrast, de novo designed proteins expand the repertoire of attributes not previously explored by natural evolution, thereby opening the door for more steerable control of protein function.
“[In de novo design], the proteins we generated have limited sequence similarity to any natural proteins,” said Arvind Pillai, PhD, one of the lead authors of the Nature paper and postdoctoral scholar in the Baker lab, in an interview with GEN. “They’re not the result of a small mutational walk from an existing protein or suturing two natural proteins together. They’re unrelated to anything that has evolved over the last 3.5 billion years.”
Specific de novo protein assemblies designed in the Nature paper included rings, formed by dimerization of two monomers, which upon assembly triggered a light output for biosensing applications, and cage-like structures, which underwent controlled disassembly for applications in releasing payload for drug delivery. These protein dynamics were experimentally validated in vitro, by size-exclusion chromatography, mass photometry, and electron microscopy.
Pillai highlighted that the ring structures exhibited additional precision properties, such as cooperativity, a phenomenon demonstrated by natural systems, such as the blood protein, hemoglobin. In cooperative systems, binding of one molecule enhances the binding of others, resulting in a rapid, switch-like response that is crucial for precise control, such as capturing oxygen in the lungs and releasing oxygen into the tissues.
“Historically in the lab, we’ve done a lot of work to control the affinity with which you can bind something, such as binding it increasingly tighter. But that’s not the only aspect that’s relevant for biological systems,” Pillai told GEN. “Sometimes you want to be able to bind in a very narrow concentration range.”
The work paves the route for engineering allosterically controlled functionalities beyond protein assembly and disassembly, such as modulating enzyme activity for metabolic function and nanomachines that can convert energy into mechanical work, similar to actin and myosin, proteins responsible for cell movement.
“The next step is to determine whether we can form interactions with small molecules and catalyze reactions accurately, which is a much more challenging frontier for the field as a whole,” Pillai said.
Looking ahead, the research team seeks to evaluate these engineered protein dynamics under broader biological context. Future work includes installing these designed functions on the surface of cells in tissue culture to provide a valuable tool for feedback control in therapeutics, such as adoptive cell therapies.
*David Baker, PhD, will be a keynote speaker during GEN’s inaugural virtual summit, “The State of AI in Drug Discovery,” scheduled to stream on October 30, 2024.
