Researchers from the Advanced Science Research Center at the CUNY Graduate Center (CUNY ASRC) designed an experiment to test whether performing X-ray crystallography imaging using elevated temperature versus elevated pressure would reveal distinct protein shapes. A better understanding of the shapes proteins take on would give researchers important insight into stopping or treating diseases.
However, current methods for revealing these dynamic, three-dimensional forms offer scientists limited information, according to the CUNY scientists, who published their paper “Pushed to extremes: distinct effects of high temperature versus pressure on the structure of STEP” in Communications Biology.
“Protein function hinges on small shifts of three-dimensional structure. Elevating temperature or pressure may provide experimentally accessible insights into such shifts, but the effects of these distinct perturbations on protein structures have not been compared in atomic detail. To quantitatively explore these two axes, we report the first pair of structures at physiological temperature versus. high pressure for the same protein, STEP (PTPN5),” wrote the investigators.
Key catalytic loops
“We show that these perturbations have distinct and surprising effects on protein volume, patterns of ordered solvent, and local backbone and side-chain conformations. This includes interactions between key catalytic loops only at physiological temperature, and a distinct conformational ensemble for another active-site loop only at high pressure. Strikingly, in torsional space, physiological temperature shifts STEP toward previously reported active-like states, while high pressure shifts it toward a previously uncharted region.
“Altogether, our work indicates that temperature and pressure are complementary, powerful, fundamental macromolecular perturbations.”
“Protein structures don’t sit still; they shift between several similar shapes much like a dancer,” said the study’s principal investigator Daniel Keedy, PhD, a professor with the CUNY ASRC’s Structural Biology Initiative and a chemistry and biochemistry professor at The City College of New York and the CUNY Graduate Center. “Unfortunately, existing approaches for viewing proteins only reveal one shape, or suggest the presence of multiple shapes without providing specific details. We wanted to see if different ways of poking at a protein could give a us a more detailed view of how it shape-shifts.”
For their experiment, the team obtained crystals of STEP, also known as PTPN5—a drug target protein for the treatment of several diseases, including Alzheimer’s—and agitated them using either high pressure (2,000 times the Earth’s atmospheric pressure) or high temperature (body temperature), both of which are different from typical crystallography experiments at atmospheric pressure and cryogenic temperature (-280 F, -173 C).
The researchers viewed the samples using X-ray crystallography and observed that high temperature and high pressure had different effects on the protein, revealing distinct shapes.
While high pressure isn’t a condition that proteins experience inside the body, Keedy said the agitation method exposed different structural states of the protein that may be relevant to its activity in human cells.
“Having the ability to use perturbations such as heat and pressure to elucidate these different states could give drug developers tools for determining how they can trap a protein in a particular shape using a small-molecule drug to diminish its function,” Keedy added.