The Advanced Photon Source
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Decades after prediction, potential applications of rippled β sheets becoming clearer

Two sets of colored graphics, one showing a singe peptide and the second showing two interleaved peptides.

One of the cool things about science is that new discoveries lead to predictions that can be tested. This leads to new experiments and, hopefully, more new discoveries. Of course, sometimes this takes longer than you might think. 

In 1953, when protein chemist Linus Pauling and X-ray crystallographer Robert Corey proposed structural elements for proteins, including α-helices and pleated β-sheets, they also predicted that another form of β-sheet was theoretically possible, the rippled β-sheet. However, it was not until recently that this structural motif was observed when peptides containing D-amino acids were mixed with peptides containing the L-amino acids that are more commonly observed in biological systems. 

Researchers predict that these rippled β-sheet structures may be useful tools for the creation of improved biomaterials, such as more stable hydrogels for drug delivery, and may have therapeutic uses in understanding or perhaps treating diseases where misfolding and aggregation of toxic peptides has been shown to be important to disease development, such as in Alzheimer’s disease and type 2 diabetes. Now, recent research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamline at 24-ID-E at the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne national Laboratory, has extended our understanding of rippled β sheet structures at the atomic level. 

The research builds on previous work that suggested, using biophysical methods, that racemic mixtures of peptides could form rippled β-sheet structures. These structures are formed by rows of peptides connected by hydrogen bonds between adjacent backbone molecules that form a sheet with the amino acid side chains sticking up out of the plane of the sheet. For a pleated β sheet, the amino acid side chains line up neatly, forming a pleat in the sheet. For a rippled β sheet, the addition of the mirror-image peptides causes the amino acid side chains to align by alternating between the D- and L- forms, forming a kind of wave or ripple along the axis of the sheet (Figure 1). 

Next, when a racemic mixture of the Amyloid β (Aβ) peptides that cause brain neuron toxicity in Alzheimer’s disease were found to generate fibrils that don’t cause the same toxic effects in neuronal model systems as when the single enantiomer peptides are used, researchers wondered about the role of rippled β sheet structures in this phenomenon. Clear structural evidence for rippled β sheets was obtained when a racemic mixture of 3-amino-acid peptides was crystallized and demonstrated the rippled structure. 

Now, the same team from the University of California has extended this research by solving the crystal structures of five racemic peptide mixtures using longer amino-acid Aβ peptides (5-7 amino acids) from AD and Amylin peptides implicated in type 2 diabetes to provide additional detailed information about the rippled β sheets formed by these peptide mixtures.

The results of the team’s analysis of the crystal structures for the racemic peptide mixtures from Aβ and Amylin confirmed that they all formed rippled β sheet structures and all formed crystals with strictly alternating D- and L- amino acids. This is different from what was observed in solution where racemic mixtures of Aβ peptides were shown to generate separate D- and L-containing structures. Interestingly, for one of the peptides, D,L-Aβ 35-40, they solved two structures in different solvents and showed that the rippled β sheet structure was maintained but the stacking of the sheets varied in terms of the amount of solvent permitted between the sheets. 

This is a new finding as all of the previous rippled β sheet structures were tightly packed “dry” structures that excluded solvent. This may have implications for understanding how to vary these structures for uses where solvent access may be a desirable characteristic of the structure, as in a drug delivery hydrogel. The other structures were all dry structures, including the diabetes peptide from Amylin (Amylin 25-29). 

Crystallographers have had decades to analyze and understand the features of pleated β sheets in protein structure. Although predicted 70 years ago, our understanding of rippled β sheets is still new and this research confirms and extends our knowledge of rippled β sheets, providing important information that may lead to new therapies and therapeutic tools.  – Sandy Field


See: A. Hazari1, M.R. Sawaya2, M. Sajimon1, N. Vlahakis2, J. Rodriguez2, D. Eisenberg2, J.A. Raskatov1 “Racemic peptides from amyloid β and amylin form rippled β-sheets rather than pleated β-sheets ,” J. Am. Chem. Soc. 2023, 145, 47, 25917-25926(November 2023)

Author affiliations: 1University of California Santa Cruz; 2University of California Los Angeles.

Dedicated to Harry Barkus Gray; A.H. and J.A.R. thank the Seaver Institute for a generous gift. We acknowledge the NIH R01AG070895, R01AG048120, RF1AG065407, and R01AG074954. We thank the NSF (MCB1616265) and the California NanoSystems Institute at the University of California, Los Angeles. The authors also acknowledge the Department of Energy Grant DE-FC02-02ER63421 for support. This work is based upon research conducted at the Northeastern Collaborative Access Team beamline 24-ID-E, which is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory, under Contract No. DE-AC02-06CH11357. We thank Duilio Cascio for support during data collection and refinement.

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