With the wide variety of sizes, shapes, and charged characteristics of proteins, no one would think there’s a simple way to categorize the molecules based on their dynamic properties. But that’s exactly what Colón has developed.
Adapting an everyday method that is used time and again in biology labs, Colón has fashioned an assay, a regimented laboratory procedure that effectively separates kinetically stable proteins from all others. In a paper published two years ago with then graduate student Marta Manning ’03, Ph.D. ’06, Colón showed that proteins that don’t easily unfold in nature were also resistant to a common laboratory treatment.
When proteins are unfolded or denatured, they can be sorted by running an electric current across a length of gel. Colón developed a simple assay on a gel that involves comparing the migration of heated and not heated protein samples containing a common chemical denaturing agent known as SDS. Most proteins migrated to the same location on the gel regardless of whether or not the sample was heated. In contrast, kinetically stable proteins exhibited a slower migration when the sample was not heated.
At first, Colón used purified proteins to make his case that kinetically stable proteins were resistant to unfolding with SDS treatment. Now, with one clever quarter turn of his gel, Colón has streamlined his procedure so that proteins, even messy mixtures of thousands of proteins, can be processed via a high-throughput assay, to identify those proteins that are kinetically stable.
With this method, he can screen the protein soup of an entire organism, as he has recently done with the bacterium E. coli, for kinetically stable proteins. Identifying the proteins that were sorted involved a series of biochemical methods and the help of Qishan Lin, director of the University at Albany’s proteomics facility.
Ultimately, Colón wants to use this method with human plasma where it might be used to diagnose medical conditions. “For example,” Colón says, “we hope to be able to identify whether any disease may be linked to the loss or gain of protein kinetic stability. The gel becomes almost like a fingerprint of kinetically stable proteins in human plasma.”
“We have now what we didn’t have before, what nobody had before: a list of kinetically stable proteins with known 3-D structures,” says Colón. “Chris is analyzing the structure of these, looking for common features that may be responsible for kinetic stability.”
Proteins that resist unfolding are a small minority in nature. “Proteins are in the stomach, in Yellowstone geysers, harsh environments like that,” says Bystroff, describing environments so harsh, it’s hard to imagine any dancing molecule surviving.
As an expert in protein unfolding, Bystroff is discovering how these proteins are kept folded. “I categorized things according to the topological features these proteins had,” he says. Categories include “tucked in,” where ends of the chain were buried like a tab, “latches,” a long piece of chain that goes around the molecule like a belt, and “blocking.”
“If you have a box that can only open one way and it’s packed up against another box,” like a hinge that can’t move, “I call that blocking,” Bystroff says.
“I’m interested in being able to design proteins,” Bystroff says. “Proteins are incredibly flexible molecules,” for which there are lots of industrial uses. To look at a protein and be able to make that protein kinetically stable, “it’s sort of natural nanotechnology.”
For all his modeling and computational expertise, Bystroff is not afraid to rely on his visual sense. “He does want to picture things,” says Mohammed Zaki, an associate professor of computer science, who has worked with Bystroff on modeling protein unfolding pathways projects. “It’s a very hard problem, a grand challenge, so one really needs all the ingenuity that we can get.”
“The good thing about Chris is he has the ability and the skills to handle both the wet lab side of things as well as the computational side of things,” says Zaki. “He’s pretty well diversified in his interests. It allows him to collaborate with Freddie on the dynamic side and with me on the data-mining side.”