Designing Large “Ideal” Proteins from Scratch

A team from Japan and the U.S. says it has identified the design principles for creating large “ideal” proteins from scratch, paving the way for the design of proteins with new biochemical functions. Their study (“Role of backbone strain in de novo design of complex α/β protein structures”) appears in Nature Communications.

The researchers had previously developed principles to design small versions of what they call “ideal proteins,” which are structures without internal energetic frustration. Such proteins are typically developed with beta strands, which serve a key structural role for the molecules. In previous designs, the researchers successfully designed alpha-beta proteins with four beta strands.

“We previously elucidated principles for designing ideal proteins with completely consistent local and non-local interactions which have enabled the design of a wide range of new αβ-proteins with four or fewer β-strands. The principles relate local backbone structures to supersecondary-structure packing arrangements of α-helices and β-strands,” write the investigators.

“Here, we test the generality of the principles by employing them to design larger proteins with five- and six- stranded β-sheets flanked by α-helices. The initial designs were monomeric in solution with high thermal stability, and the nuclear magnetic resonance (NMR) structure of one was close to the design model, but for two others the order of strands in the β-sheet was swapped. Investigation into the origins of this strand swapping suggested that the global structures of the design models were more strained than the NMR structures.

“We incorporated explicit consideration of global backbone strain into the design methodology and succeeded in designing proteins with the intended unswapped strand arrangements. These results illustrate the value of experimental structure determination in guiding improvement of de novo design, and the importance of consistency between local, supersecondary, and global tertiary interactions in determining protein topology. The augmented set of principles should inform the design of larger functional proteins.”

Potential applications

Engineered proteins would be useful for drug and vaccine development, especially for formidable viruses like HIV or rapidly changing ones, like SARS-CoV-2 and the flu. Proteins designed to exact specifications might also prove therapeutically useful in cleaving mutated genes, and for speeding up chemical reactions important in industry and environmental reclamation.

However, while the team found that the designed proteins were structurally ideal, they are too small to harbor functional sites.

“We set out to test the generality of the design principles we developed previously by applying them to the design of larger alpha-beta proteins with five and six beta strands,” said co-first author Nobuyasu Koga, PhD, associate professor in the Institute for Molecular Science of NINS.

The results were puzzling. They found that their experimental structures differed from their computer models, resulting in proteins that folded differently by swapping the internal locations of their beta strands. The team struggled with the strand swapping puzzle, but by iterating between computational design and laboratory experiments, they reached a conclusion.

“We emphasize that experimental structure determination is important for iterative improvement of computational protein design,” said co-first author Gaohua Liu, PhD, chief scientific officer of Nexomics Biosciences.

“Sometimes we learn the most from these ideal proteins when their experimental structures differ, rather than match, their intended design, since this can lead to a deeper understanding of the underlying principles,” added Gaetano Montelione, PhD, co-author and professor of chemistry and chemical biology at Rensselaer Polytechnic Institute.

The reason for the strand swapping, they determined, was due to the strain of the whole system on the foundational backbone structure. According to Nobuyasu Koga, PhD, the strain is global, instead of connection to connection. Proteins can adjust the length and register of strands across the system to alleviate this backbone strain.

Next, the researchers plan to continue studying the trade-off between more functional proteins with what could be considered less-than-ideal qualities.

“We would like to design proteins with more complex functional sites by incorporating non-ideal features such as longer loops, which are important not only for function but also for relieving global backbone strain,” said David Baker, PhD, co-author and professor of biochemistry at the University of Washington.