By TOM DINKI
Published September 19, 2023
Unlike an athlete or musician under pressure, most proteins must fold to perform at their best.
These large biological molecules fold, bend and twist into unique conformations in order to carry out their various functions, like transmitting signals between cells and repairing tissues.
The conventional wisdom is that proteins unfold — or denature — at high temperatures, as the heat causes atoms to move so rapidly that they lose their structure. Far less understood is why proteins destabilize and may even unfold at low temperatures.
“It’s a phenomenon that is kind of counterintuitive: Why should a protein get less stable or even unfold if you cool it to very low temperatures? It should stabilize, but it generally doesn’t,” says Thomas Szyperski, UB Distinguished Professor of Chemistry, College of Arts and Sciences. “This is because of thermodynamically and structurally quite complex interactions between protein and water at low temperatures. Very cold water — supercooled below the freezing point — acts like a protein denaturant.”
Szyperski and senior staff scientist Surya Venkata Pulavarti have, for nearly a decade, researched cold unfolding proteins, as this could lead to a better understanding of life in extremely cold ecosystems, as well as assist protein design and drug development that requires cool temperatures.
They will continue that work — and explore new questions, like the role of pressure on cold unfolding — with a new $710,128 collaborative grant from the National Science Foundation (NSF). Szyperski’s longtime collaborator Brian Kuhlman, protein designer and professor of biochemistry and biophysics at the University of North Carolina at Chapel Hill, has received $206,912 for the project.
As a result of the funding, Szyperski and Kuhlman, who are both principal investigators on this collaborative grant, can continue to apply for access to national resources. This includes NSF’s Anton 2 supercomputer at the Pittsburgh Supercomputing Center, which was designed to dramatically increase the speed of molecular dynamics simulations; NSF’s ACCESS system of supercomputing facilities; and Stanford University’s Synchrotron Radiation Light Source, which allows for small-angle X-ray imaging of proteins in solution.
“We are excited about our ongoing collaboration with the Szyperski lab, as it allows us to use recent developments in computational protein design to probe fundamental questions in protein folding and stability,” Kuhlman says.
Research collaborators include Edward Snell, an X-ray crystallographer and professor in the UB Department of Materials Design and Innovation, and CEO of the Hauptman-Woodward Medical Research Institute; Van Ngo, an expert in molecular dynamics simulations and senior scientist at Oak Ridge National Laboratory; Catherine Royer, an expert in high-pressure protein unfolding and professor of biological sciences at Rensselaer Polytechnic Institute; Joan-Emma Shea, an expert in molecular dynamics simulations and professor of chemistry at the University of California Santa Barbara; and Thomas Weiss, an expert in small-angle X-ray solution scattering and senior staff scientist at the Stanford Linear Accelerator Center at Stanford University.
“It is one of the blessings of this project to be able to work with such an exceptional group of world-renowned scientists. This also provides unique opportunities for students joining us,” Pulavarti says.” I am delighted to pursue this highly interdisciplinary project using a very broad range of cutting-edge computational and biophysical techniques.”
One reason scientists know so little about cold unfolding in comparison to heat unfolding is that it’s difficult to observe in a lab setting.
“You need to study the proteins at temperatures well below the freezing point of water,” Szyperski says. “Most naturally occurring proteins simply don’t cold unfold in a temperature range where you could study the phenomenon and they do not feature a molecular core that is suitable to test our hypotheses related to cold unfolding.”
A method of protein design — known as de novo — allows scientists to create novel proteins that don’t occur naturally. They can then observe cold unfolding in the temperature range suitable for biophysical characterization and to specifically test hypotheses. In other words, the proteins are designed to yield unambiguous experimental answers.
“With de novo protein design, you are killing two birds with one stone,” says Szyperski, adding that he and his collaborators are thus far the only scientists using de novo protein design to study cold unfolding.
Since this work is practically uncharted territory, Szyperski says their efforts can simultaneously improve computational design protocols.
“This benefits the broader protein design community,” he adds.
Supported by prior NSF funding, Szyperski and Kuhlman used the technique to shed new light on cold unfolding in a study published in the Journal of Physical Chemistry last year.
Placing designed proteins into capillaries and cooling them below zero degrees Celsius, they found that cold unfolding is initiated when water infiltrates the protein’s de novo designed, partially hydrophilic core. The hydrophilic segment acts as an entry point for water, while cold unfolding proceeds when the hydrophobic segment is also exposed to water.
In the new project, they will not only profile stability by temperature, they’ll also profile by pressure of up to 3,000 atmospheres.
“That provides a plethora of new information,” Szyperski explains. “A very large fraction of the ecosystems on our planet actually exists at very low temperatures below freezing, like the Arctic, or at very high pressure of up to 1,000 kilobars, like in the deep sea. So, being able to understand how these protein molecules react to low temperatures and high pressure is crucial.”
The research is also important for protein drugs and their formulation. Protein drugs must be kept at low temperatures to remain stable and active, so it’s valuable to know the temperature and conditions at which specific proteins are destabilized or even cold denature. Moreover, the research may impact the engineering of novel cold-adapted enzymes, important targets in biotechnology.
Some of the collaborators are involved in Szyperski and Pulavarti’s other project, the NSF Science and Technology Center BioXFEL. Directed by Snell, headquartered in Buffalo and led by UB, BioXFEL uses X-ray-free electron lasers to understand how life works at the molecular level.
Through BioXFEL, Szyperski, Snell and Weiss, as well as Marius Schmidt, professor of physics at the University of Wisconsin-Milwaukee, are currently studying how enzymes change shape during substrate turnover.
“We are looking at the protein while it’s in action, and we are learning a lot about what’s happening when the proteins turn over,” Szyperski says. “We’ve gotten a couple unprecedented insights. There is synergy between the two projects.”
As for the cold unfolding project, Szyperski and his team will test their hypothesis that cold unfolding — even when done only partially and infrequently — is important to overall protein function.
Szyperski says these partially cold unfolded conformations — which are lowly populated states at ambient conditions — may be functionally important, which scientists don’t currently realize.
“These partially cold unfolded conformations, which are away from the folded state, may in some cases actually be the ones driving function,” he says.