Imaging Proteins to Understand Their Function
Melody Campbell, Structural Biologist
Our bodies are primarily made up of water, fat, and proteins — all of which we must regularly consume to make new cells and maintain the ones we have. When we eat our favorite snack, our body will break down the protein molecules it contains into their basic components. It will then use those parts to synthesize the building blocks for making new cells, including amino acids which will go on to become the proteins that our body and cells need to function. Every cell in our body contains millions of proteins, each having thousands of possible forms and functions. Proteins are responsible for just about everything our cells do, including transporting nutrients, neural communication and sensation, cell division, protecting us from invading pathogens, moving our muscles, and so much more.
Proteins are made from “chains” of linked-up amino acids which fold into three-dimensional structures. It’s this structure that determines how a protein interacts with other molecules and dictates its function. A protein’s structure gives investigators clues to its function. And in the event that a protein is mutated, scientists can use the structure to figure out why the protein stopped working. Even swapping out a single amino acid for another can dramatically change a protein’s shape and how it works.
Understanding protein function is essential to overcoming just about every health challenge humanity has faced, from genetic disorders to cancer and infectious diseases. This need is what drives Dr. Melody Campbell to uncover the structure of proteins, in order to provide a better understanding of how they interact with other molecules, how they work — and how they stop working.
Campbell’s ever-present interest in the world around us stretches as far back as she can remember.
“As a girl, I was interested in everything,” she said. “Understanding how things work has always been a big part of what I like to do.”
This enthusiasm carried through to college where she explored many interests, including chemistry and music. This exploratory drive is what led her to join a research lab, and like many eventual scientists, how she found herself spending her first summer washing laboratory glassware.
“My advisor noticed how efficient I was at washing dishes,” she said. “I would finish quickly enough that I started being able to help with research projects.”
Having proven her chops at scrubbing beakers and showing an aptitude for doing experiments, she was soon given the opportunity to start her own research project.
“I really liked research because I could ask a question that no one knew the answer to and go find an answer myself,” she said.
Despite enjoying working in a lab, Campbell hadn’t really considered a career as a scientist. When deciding on a path, she ultimately settled on pharmacy as a sensible option she expected would lead to a stable job. While wrapping up one of her research projects and preparing to enter pharmacy school, her senior-year lab advisor called Campbell into her office and shut the door.
“That's usually never a good sign,” Campbell recalled thinking. “I thought I had broken something!
But instead, Campbell’s advisor thought she could have a promising career in science and suggested applying to graduate school. Campbell took her offer to join the lab as a technician while she considered whether to pursue pharmacy or research. It was during this time that Campbell first encountered a research technique called cryogenic electron microscopy, or cryo-EM. This technique allows researchers to use high-powered microscopes to take detailed pictures of proteins to better understand their structure and ultimately their function. Campbell didn’t know it at the time, but cryo-EM would go on to define her career.
“No idea seems too big or too unattainable.”
Until encountering cryo-EM, Campbell’s day-to-day research activities had consisted of performing biochemical assays on proteins — which involved moving a lot of tubes of liquid. She still vividly remembers the first time she saw an image of a protein she was studying.
“We had started collaborating with a lab doing cryo-EM and I would give them some proteins in a small tube that just looked like a clear liquid. They would then send back an email with an actual structure, like a map that you can visually see,” she said. “It's no longer the interpretation of a protein through charts and graphs. You're literally seeing the shape of what the protein looks like!”
Campbell made up her mind right there: She wanted to go to graduate school and become a structural biologist. But at the time, other scientists dismissed cryo-EM as “blobology,” because the resolution of its images was so low.
“Using cryo-EM to image proteins was like looking at the earliest televisions that were black and white and very grainy,” she said.
But despite its low resolution, cryo-EM allowed scientists to visualize proteins that couldn’t be imaged using standard techniques. Campbell saw the technology's potential and wanted to be at the forefront of both using cryo-EM and developing methods to improve it. Her timing couldn’t have been better. The cryo-EM “resolution revolution,” when the technique’s visual clarity improved dramatically, was just beginning. In graduate school, she helped develop new and improved algorithms that dramatically enhanced the resolution of images created using cryo-EM.
One of the challenges of improving the resolution of structures captured with cryo-EM was tied to the microscope itself. To perform cryo-EM, scientists use transmission electron microscopes, which produce images by passing a beam of electrons — not visible light — through biological specimens. But proteins are so small that the high-energy electrons cause them to move and blur the image. The trick to boosting the clarity of the cryo-EM picture was to take a sequence of images and develop software to unblur it.
“You can write software to realign the proteins in all of those frames to each other so you can get a really clear picture,” Campbell said. “Over the course of my Ph.D., I continued to develop this technology while trying to apply it to new and more complicated proteins. Now, the most widely used cryo-EM software is based on the initial methods that we developed.”
These improvements have made it possible for researchers to visualize how individual molecules interact with proteins — a crucial threshold for designing therapeutics that target specific parts of proteins.
While doing her postdoctoral research, Campbell began using her cryo-EM expertise to study integrins, a family of proteins on which she’s built her research program. The many vital activities in which integrins play a role include communication between cells, cell growth and proliferation, cellular movement and immune function. Changes to integrin structure and function can lead to numerous disorders, including autoimmune, cardiac, pulmonary and blood diseases as well as cancer and increased susceptibility to infections. The involvement of integrins in a variety of crucial functions is precisely why Campbell finds them so fascinating.
In particular, she’s interested in a family of integrins found on white blood cells, immune cells that are one of our first lines of defense against invading pathogens. White blood cells move through the body, find pathogens and eliminate them. Integrins are essential to all these processes. Despite the importance of integrins to our health, there remain many questions about how, exactly, they work.
Understanding the structure of integrins will allow researchers to better understand how they function — but historically integrins have posed a lot of challenges to structural biologists. Before the advent of cryo-EM, alternative imaging techniques limited researchers to studying an integrin one fragment at a time. Cryo-EM allowed scientists to image the whole protein, which gives more information about how each of its parts work together. But the approach wasn’t perfect.
“When I first started, integrin proteins were considered too small and too flexible to study at high-resolution using cryo-EM,” Campbell said.
Now, thanks to improvements in software and analysis (many of which Campbell helped develop), it is possible to create finely detailed pictures of integrins that allow researchers to identify regions that are critical for their interactions with other proteins. It’s a far cry from where Campbell started out.
Back then, “We collected images one at a time on physical film, which had to be developed in a dark room, scanned, and finally processed computationally — a procedure that took months or years,” she recalled. “Now, we have solved, highly detailed structures in just a couple of hours. These advances in both speed and resolution have enabled us to answer questions that before would have been impossible. No idea seems too big or too unattainable.”
— By Matthew Ross, June 13, 2022
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