A hairpin twist hints at nature’s answer to a long-running molecular mystery

Written by
Stephanie Pappas, for the Office of Engineering Communications
Aug. 18, 2022

A substance long reviled as a problematic research byproduct turns out to play a key role in the development of some peptides, according to a new Princeton Engineering study.

The naturally occurring chemical group, called aspartimide, is usually found interfering with cancer therapies and spoiling biological experiments. But a paper in Nature Chemistry, published Aug. 18, shows that aspartimide also provides a key structural link in some peptides, tiny protein-like molecules at the center of many modern medicines.

Researchers from A. James Link’s lab noticed aspartimide’s structural role in a newly discovered peptide with an unusual shape. Rather than forming a helix or a strand, like most known peptides, this one curves back on itself into a hairpin shape. Even more unusually, the two sides of the hairpin are cross-linked by two esters, acting like rungs on a ladder and adding mechanical strength to the structure. The aspartimide group is found in the loop of the hairpin.

“We’re still trying to figure out what that actually means for biological function,” said Link, a professor of chemical and biological engineering. He said the ultimate goal of the research is to identify natural products with novel functions, some of which could be used to design new drugs. But along the way he relishes findings like this, which presents new starting points for investigation and overturns years of assumptions about a well-established problem.

researcher's gloved hand lifts a vial from an array of vials in a compartment

Researchers from Link's lab discovered an unusual gene cluster in one bacterium, then extracted those genes and placed them in E. Coli, which is easier to work with in lab settings. The E. Coli produced the strange hairpin peptide with its structurally important aspartimide ring. Photo by Sameer A. Khan/Fotobuddy

“Usually, people think of aspartimides as kind of like weeds in your garden. They’re a side product and a nuisance when you do peptide synthesis,” said Hader Elashal, a senior scientist at NJ Biopharmaceuticals who was first author of the new paper while a postdoctoral scholar in Link’s lab. “In this case, you actually want the weeds. They’re growing for a purpose.”

The world of biomolecules is easily as diverse as a deep-sea thermal vent or a rainforest bursting with insects. It’s also just as mysterious. By studying the natural products of organisms like compost-dwelling bacteria, Link and his team discover never-before-seen chemical structures and the proteins that put those structures together.

The team hunted through the genome of one such decomposer and found an odd gene, the equivalent of a neon sign pointing to the blueprint for a mystery peptide. They lifted the gene and its neighbors and plugged them into E. coli, a bacterium that grows readily in lab environments. The E. coli pumped out the new hairpin peptide.

They found that the hairpin is made of a chain of 21 amino acids. It gets its shape from a series of cross-linkages that bind amino acids on the far ends of the chain. These cross-linkages protect the peptide from getting degraded by enzymes that would break it down, Link said. That structure may provide some clue to its function in the wild, but Link said more study will be needed to uncover those ties.

The most exciting finding was on the bend of the hairpin, where that odd gene first noticed by Link’s team (the neon sign) builds a specialized chemical group. That protein adds a cluster of hydrogens and carbon to oxygen, ultimately making a 5-membered ring – the aspartimide.

The researchers call the new peptide fuscimiditide. They conducted experiments on the pre- and post-aspartimide hairpin and found that the little ring makes the peptide much more rigid. It also changes the charge of the peptide from negative to neutral. That might be important, Link said, because cell membranes are negatively charged. Thus, a negatively charged peptide would never be able to interact with a cell membrane. The two would repel each other like head-to-head magnets.

If the aspartimide does enable the peptide to interact with cell membranes – and that’s a big if – it could be useful information for synthetic biologists. “One big area of research is cell-penetrating peptides,” Link said. “You could use a peptide to be able to get into a cell and then conjugate that to a cancer drug or something similar.”

But that’s still far off. Right now, Link and his team are focused on learning more about the chemistry of this elegant little peptide. They’ve managed to synthesize it from pure materials in a test tube, allowing an unprecedented look into its construction.

“We want to ultimately find new drugs,” Link says, “but along that path, if we can find new chemistry, new shapes, new enzymes, that’s something that gets me very excited.”

The paper "Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide" was published with support from the National Institutes of Health, the National Science Foundation and Princeton University's School of Engineering and Applied Science. In addition to Elashal and Link, authors include Wai Ling Cheung-Lee, Brian Choi, Li Cao, Joseph D. Koos, Michelle A. Richardson and Heather L. White.