Proteins are the heavyweights in biochemistry. These beef molecules function as building blocks, receptors, processors, couriers, and catalysts. “Proteins are the molecular machines that power all life on Earth,” explained Mark Sherwin, professor of physics at UC Santa Barbara. Of course, scientists have devoted a lot of research to understanding and manipulating proteins.
A team led by researchers at UC Santa Barbara, including Sherwin, has made strides in tackling one of the grand challenges of modern science: recording proteins in motion in a realistic environment. The authors discuss their technique in Angewandte Chemie, a journal of the German Chemical Society. The approach could revolutionize our understanding of how proteins do their jobs and guide the design of proteins for specific purposes.
A daunting challenge
Understanding the function of a protein requires more than a simple list of its parts. For these molecules, form begets function. Scientists have made tremendous progress in the past 20 years deciphering the shapes of proteins based on the amino acid building blocks that make them up.
However, even seeing the shape of a machine often is not enough to understand how it works. “Imagine you’re an alien and you see an image of a sewing machine,” Sherwin said. “You’d be hard-pressed to understand what he’s doing. But if you saw a movie, you’d have a much better idea.”
Unfortunately, that’s a tall order for protein. Although they are relatively large molecules, proteins are still only a few nanometers in size, 100 times smaller than we can resolve with even the most powerful optical microscopes. And they exist in wet, cramped environments that are not conducive to cinema.
“One of the biggest challenges in biology in general is to see proteins in action,” explained co-lead author Shiny Maity, a PhD student in chemistry. It is much easier for scientists to examine the structure of proteins when they are frozen. Seeing them move requires a technique like stop-motion animation: start the action, freeze the protein, take a picture, repeat. This is often prohibitively difficult for both fast and slow motion. In addition, rapid freezing of the protein can affect its structure.
“Our goal is to completely eliminate the freezing aspect and see protein movement in as vivid an environment as we can get,” said co-lead author Brad Price, a graduate student in physics.
A complicated technique
This article presents a new method for monitoring the movement of proteins in a realistic environment after their movement has been triggered by an external event (in this case, a pulse of visible light). The authors call the technique TiGGER, for time-resolved gadolinium-gadolinium electron paramagnetic resonance. It is elaborate, requiring quantum effects, specialized chemistry, specialized equipment and industrial engineering.
TiGGER involves labeling two points on the protein and tracking the distance between those labels as the protein unfolds and refolds. The star of the show is a charged gadolinium atom or ion. Its electrons align in such a way that the ion behaves like a small magnet. If you place it in a strong magnetic field, it will align with or against the external field and begin to oscillate.
The scientists glue the gadolinium into a molecular cage to stabilize it and add some chemical scaffolding to attach it to the protein. But these pieces are attached to only one type of amino acid, cysteine. So the team had to change the amino acids they wanted to add to cysteines without affecting the overall function of the protein. It was a task made even more difficult by a cysteine in the center of the protein that is critical to its function.
“The spin label is chosen very strategically,” Maity said. “It’s big enough not to get into the core of the protein, where the functional cysteine is. But it’s also not too big to disrupt the protein’s natural shape.”
The gadolinium ion’s wobble or “transition” is affected by the proximity of the other tag, which has its own wobbled gadolinium ion that creates its own small magnetic field. This transition changes depending on how close the two tags are to each other. Measure this swing and you can derive the distance.
This is exactly what the authors did using a laser with light at energies slightly higher than those in a microwave oven. When the frequency of these sub-Terahertz waves and the transition of the ion match, the waves are absorbed. The scientists then measured this absorption to detect small changes in gadolinium transition. If the amount of absorbance changes with time, this means that the tags are moving.
Add some more math and the authors could tell you how far the tags are from each other. “We know we can get distance as a function of time, but it will take more development,” Price said.
An illuminating protein
The authors chose a popular and versatile protein to develop TiGGER. Their model belongs to the light-, oxygen-, or voltage-sensitive (LOV) family of proteins, specifically a light-activated protein called AsLOV2. “LOV proteins control processes ranging from circadian rhythms in bacteria, plants and fungi to phototropism in plants and microorganisms,” said co-author Max Wilson, assistant professor in the Department of Molecular, Cell and Developmental Biology. “In short, they are closely related to the sense of light.”
This property makes AsLOV2 popular with scientists and engineers, as well as simple to use. “It’s interesting and a perfect test case,” Price said, “a best-of-both-worlds situation.”
LOV proteins allow scientists to use light as a “remote control” for a whole range of molecular processes in cells. “We use it to control stem cell differentiation, antibody binding, the stiffening and loosening of extracellular matrix proteins, and the activation of cell signaling pathways,” Wilson said.
Assistant Professor Arnab Mukherjee, of the department of chemical engineering, uses LOV proteins to monitor biochemical processes in living cells using fluorescence, like a highlighter under a black light. “Unlike conventional fluorescent proteins, LOV proteins work by a distinct mechanism that makes their ‘glow’ visible even in oxygen-free conditions,” he explained. This offers a tool for studying microbes that live in anaerobic environments, such as the human gut.
But engineering these proteins to do what researchers want them to do is difficult. This is where TiGGER comes in handy. If scientists like Wilson and Mukherjee can see proteins in motion, they could be more careful in their design processes.
A look to the future
Senior authors Sherwin and Songi Han, professor of chemistry, first began their effort to film proteins in 2006, but it’s still early days for TiGGER. Currently, the technique can produce a one-dimensional trajectory of a protein’s movement between two points. But its real power comes from repeating the technique in many different locations. This enables scientists to piece together the movement of the protein as a whole. They can then map that movement onto a model of the protein to create a movie in a similar way to the CGI animation that brings our favorite cartoon characters to life.
The authors are focused on optimizing the technique before spending time applying it to other sites in AsLOV2. They are working to increase the signal-to-noise ratio and increase the sampling speed of their instruments. The team also hopes to slow down the random movement of the proteins as they float in solution, which will allow them to take sharper shots than they can now.
In the meantime, Price and Maity use TiGGER to answer some basic questions about AsLOV2. For example, why does the protein unfold more than 1,000 times faster than it folds? And how do mutations known to affect folding affect unfolding? They are also investigating how warmer conditions affect protein function. The results could shed light on how oats – the source of AsLOV2 – will respond to climate change.
Ultimately, TiGGER can be translated to all kinds of other proteins, as long as scientists can modify the cysteine amino acid sites of interest without affecting the protein’s function. “Biophysicists try to ‘film’ proteins in motion to gain an in-depth understanding of their biological functions,” said Maity. “TiGGER has the potential to make this dream a reality.”