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Scientists uncovered the structure of the key protein for a future hepatitis C vaccine – here's how they did it

Alba Torrents de la Peña, Postdoctoral Fellow in Integrative Structural and Computational Biology, The Scripps Research Institute and Lisa Eshun-Wilson, Postdoctoral Scholar in Molecular and Cell Biology, The Scripps Research Institute, The Conversation on

Published in Science & Technology News

Cryo-EM, or cryo-electron microscopy, is a type of imaging technique that views specimens at cryogenic temperatures, in this case the boiling point of nitrogen: minus 320.8 degrees Fahrenheit (minus 196 Celsius). With temperatures that cold, ice freezes so quickly that it doesn’t have time to crystallize. That creates a beautiful glasslike frame around the protein of interest, allowing an unhindered view of every structural detail. Cryo-EM also requires very little protein to work, reducing the amount of material we would need to purify.

Winner of the 2017 Nobel Prize in chemistry and Nature magazine’s 2015 “Method of the Year” award, cryo-EM is superb for imaging biological macromolecules in their native, or natural, state in the aqueous environment of human blood. Cryo-EM was also pivotal for characterizing the structure of the COVID-19 virus and its variants.

So how do you take a picture of a protein?

First, we embedded the genetic code to make E1E2 in human cells in a petri dish so we would have sufficient amounts of protein to study. After purifying the protein, we plunged it into liquid ethane followed by liquid nitrogen. Liquid ethane is used to freeze the protein because it has a higher boiling point than liquid nitrogen. This means it is able to capture more heat before turning to a gas, allowing the protein to freeze much more quickly than it would in liquid nitrogen and avoid structural damage.

Once the protein was vitrified, or in a glasslike ice state, we were able not just to see its overall structure, but also to capture multiple individual configurations of the protein that it takes when it shape-shifts, including its less stable forms.

At this point, our protein was ready for its close-up. We employed a microscope that uses a beam of focused, high energy electrons and a very fancy camera that detects how the elections bounce off the protein’s surface. This created a 2D image that we then mathematically transformed into a 3D model. And that was how we got the coveted “close-up” of HCV’s surface protein.

Our next step was then to assess the location of each amino acid, or building block of the protein, in 3D space. Because every amino acid has a unique shape, we used a computer program that could identify each one in our 3D map. This allowed us to manually reconstruct a high-resolution model of the protein, one building block at a time.

Our 3D map and model of the HCV E1E2 protein supports previous research describing its structure while providing new insights into features that will help pave the way for a long-sought vaccine design against this virus.

 

For example, our structure reveals that the interface between the two main parts of the protein is stabilized by sugars and hydrophobic patches, or areas that push out water molecules. This creates sticky binding hubs along the protein and keeps it from falling apart – a potential site for protective antibodies and new drugs to target.

Researchers now have the tools to design antiviral drugs and vaccines against HCV infection.

This article is republished from The Conversation, an independent nonprofit news site dedicated to sharing ideas from academic experts. It was written by: Lisa Eshun-Wilson, The Scripps Research Institute and Alba Torrents de la Peña, The Scripps Research Institute. If you found it interesting, you could subscribe to our weekly newsletter.

Read more:
Chilled proteins and 3-D images: The cryo-electron microscopy technology that just won a Nobel Prize

New AI technique identifies dead cells under the microscope 100 times faster than people can – potentially accelerating research on neurodegenerative diseases like Alzheimer’s

Lisa Eshun-Wilson receives funding from the National Science Foundation.

Alba Torrents de la Peña receives funding from Netherlands Organization for Scientific Research (NWO) Rubicon Grant 45219118.


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