The crystalline sponge method makes beer delicious!?

Beer is a drink that is loved all over the world and its refreshing bitterness is appreciated by many. Surprisingly however, the bitterness components contained in beer have not yet been completely elucidated scientifically. The bitter taste of beer originates from α-acid contained in the ingredient hop. During the brewing process, α-acid is isomerized to iso-α-acid, and it wasn’t until 2013 that the correct structures of these compounds were elucidated. This example illustrates the extreme difficulty of natural product structural analysis. It is known that α-acid and iso-α-acid are further chemically transformed into various compounds, but there are very few examples solving those structures. Thus there are still many compounds contained in beer that have not been chemically revealed. For the first time in the world we have succeeded in comprehensively elucidating the structure of trace amounts of unknown bitterness compounds by effectively combining HPLC and the crystalline sponge method. The components that were formed from iso-α-acid were found to possess complicated three-dimensional structures, which is where the crystalline sponge method demonstrated its effectiveness. By elucidating the structure of these reaction products, it becomes possible to understand the mechanism of the chemical change occurring. These findings enable control over the composition of bitterness components making it possible to control the reaction to adjust the taste and maintain freshness, leading to the development of new brewing technologies. This is an important achievement, we believe, in terms of the industrial applicability of the crystalline sponge method.

The extended sponge method – Aiming for protein structural determination through encapsulation in a gigantic cage

In this article, we used a self-assembled cage complex with a giant hollow inner space to precisely encapsulate a natural protein and analyzed its structure and properties in detail for the first time. We discovered that the protein is stabilized by confinement in its inner space and that refolding of the caged proteins can be induced, repairing the three-dimensional structure from a partially denatured state. By utilizing these “encapsulation effects” we will develop a method to elucidate protein structures difficult to observe by the conventional techniques.
“Does the behavior of proteins change by confining them into a narrow space?” To answer this question that has been raised out of curiosity in the study of self-assembly, we have spent the last 10 years studying the encapsulation of proteins in the hollow inner space of artificial molecules. Proteins (molecular weight >10,000) are significantly larger than small organic molecules (molecular weight <500), making it extremely difficult to encapsulate them in the hollow complex while maintaining their three-dimensional structure. Although we succeeded in constructing the world’s largest complex molecule with an inner space large enough to encapsulate a protein, what we could do at first was to just confirm the encapsulation in the complex by adding modification to a part of the protein (Nat. Commun. 2012, 3, 1093.).
In our research published in Chem, we achieved the encapsulation of a natural protein into the hollow complex without protein engineering by developing a new method. Through the selective condensation with the protein’s N-terminus, we encapsulated a protein retaining its native structure. What is more, we evaluated the structure and enzymatic activity of the encapsulated protein and elucidated the behavior of a protein confined in a narrow space for the first time. Since in our study we use the N-terminus, which is common to proteins, this research can be applied to almost all the natural proteins.
When we encapsulated a protein called cutinase-like enzyme (CLE) and analyzed its structure and function, an astounding stabilization of the protein was observed. For instance, in acetonitrile CLE is completely deactivated and loses its enzymatic function within one hour. In contrast, by encapsulation into the cage, the activity was retained even after one month (1000-fold stabilization). We found out that this is because in denaturing acetonitrile, the protein loses its higher-order structure, resulting in aggregation, whereas the encapsulated protein is spatially isolated, thus suppressing denaturation and aggregation. To our surprise, although the structure of the encapsulated CLE started to collapse after two weeks in acetonitrile, upon changing the solvent to water its higher-order three-dimensional structure was restored and its enzymatic activity regenerated. This refolding, which is reminiscent of molecular chaperones, occurred because we encapsulated just one protein molecule in the narrow space of the cage. In this way, it became clear that even for proteins “encapsulation effect” can take place, which triggers unique properties of proteins in a narrow space.
We call this protein encapsulation chemistry the “extended sponge method” and are continuing our research with the aim of developing it into a protein structure analysis technique. The defined inner cavity of the hollow complex provides a suitable place for obtaining proteins’ detailed structural information. What is more, we believe that by utilizing the “encapsulation effect,” it is possible to capture protein structures that no one was able to observe before. For instance, by capturing unstable proteins or intermediate states inside the hollow complexes, we can elucidate dynamic and transient protein structures that were difficult to observe until now. In the future, we envision developing a technique, a completely new approach to protein structural analysis that will innovate life science.

Crystalline Sponge method: Principle and proof-of-concept studies

This review describes the historical background and principles behind the birth of the crystalline sponge method, which is our core technology. In addition, the review touches on the technical improvements found by us and researchers in Japan and overseas since the development of the crystalline sponge method, and examples of effective application research of the crystalline sponge method.