By: Stephanie Blaszczyk
How do energetic mountains and molehills affect the folding and function of proteins?
The Cavagnero Lab asked this question when investigating why bacterial proteins fold properly instead of misfolding and aggregating under physiological conditions.
“If proteins in our bodies and in bacteria were governed by the laws of thermodynamics alone, they would aggregate hopelessly, but they don’t,” Silvia Cavagnero, professor of chemistry at the University of Wisconsin-Madison, said. “To better understand why life works the way it does, we asked why.”
In a recent paper, the Cavagnero group determined that there is a kinetic obstacle – a metaphorical mountain – that acts as a natural barrier to keep proteins in their native states and prevent aggregation into toxic conformations (J. Phys. Chem. B 2018,DOI: 10.1021/acs.jpcb.8b05360).
“For thermodynamic and concentration reasons, proteins have a natural tendency to form aggregates that aren’t bioactive, and we determined that this natural tendency is blocked within healthy cells,” Cavagnero explained.
While proteins are frequently thought about in biological contexts, their three-dimensional structure often results from an inherent struggle between thermodynamic stability and kinetic effects. For more than sixty years, the Anfinsen hypothesis, which states that a protein’s amino acid sequence determines its three-dimensional structure and this structure determines its biological activity, has promoted the idea that protein conformations favor their native states because it’s the most thermodynamically favorable state. The Cavagnero group demonstrated that kinetic factors are also responsible for retaining the majority of bacterial proteins in their native conformations, which makes a noteworthy expansion to the Anfinsen hypothesis.
“It is known that protein aggregation in living cells can be deleterious and is often associated with deadly maladies like Alzheimer’s, Creutzfeld-Jakob and Parkinson’s disease,” Angela Varela, lead author on the publication and former Cavagnero group member, said. “Therefore, understanding how living cells stay healthy by avoiding aggregation is crucial.”
Rather than focusing on what makes a protein misfold and aggregate, Varela and her colleagues questioned what keeps proteins in their native, functional state: their answer – kinetic trapping, a process by which proteins are confined to non-aggregated conformations because of an energy barrier that cannot be quickly overcome without forcing conditions or extended timescales.
Think of kinetic trapping like this: On any given day, people can easily walk over a small molehill from a sidewalk. Similarly, under physiological conditions, proteins have sufficient energy to interconvert between the native, folded and unfolded states (black dotted lines, Figure 1). However, without any preparation or supplies, people would have a more difficult time quickly climbing over a mountain. Likewise, proteins do not interconvert from the unfolded state to the aggregated state during biologically relevant timescales (red dotted lines, Figure 1).
The authors discovered this kinetic trapping phenomenon by using a cyclic perturbation method where they repeatedly varied the temperature and pH of solutions containing a model protein, apomyoglobin (apoMb). Under physiologically-relevant conditions, kinetic trapping forces proteins to remain in their native folded and unfolded states. Only when the protein is subjected to non-physiologically-relevant conditions, including excessive heat or denaturing chemicals, are aggregated states observed.
The authors then wondered whether kinetic trapping could be a general phenomenon experienced by proteins other than apoMb. To probe this question, they applied a simplified cyclic perturbation approach to the soluble proteome of E. coli. They observed that most of the soluble E. coli proteome is kinetically trapped with respect to aggregated states, suggesting a variety of bacterial proteins exhibit this phenomenon.
For optimal cellular function, the cell must express thousands of proteins at high total concentrations. However, under common experimental conditions, proteins tend to aggregate if overexpressed. One major goal of biology is to understand how cells evolved to avoid aggregation while saturated with proteins. Researchers would like to harness these adaptations to allow the soluble, properly-folded expression of any protein sequence in the lab. The discovery of kinetic trapping is a step toward achieving this goal.
“Science is a collaborative process,” explained Matthew Dalphin, a co-author on the publication and graduate student researcher in the Cavagnero group. “Developing a solid understanding of how protein folding works in the cell is very important. As we learn the basic science behind how cells guide, and trap, proteins into their properly folded structures needed for biological activity, we find knowledge that may aid the future development of treatments to correct certain aggregation-based diseases.”
Researchers have long known that proteins have a natural tendency to aggregate. But the discovery that kinetic trapping dramatically slows down this aggregation at physiological conditions reveals one of the most ingenuous tricks adopted by Mother Nature to ensure proper cell function and allow life to flourish.
This work was funded by the National Science Foundation (NSF) grants MCB-0951209 and MCB-1616459 (to S.C.), including NSF REU supplements (to J.F.L.). A.E.V. was the recipient of an NSF Graduate Research Fellowship.
This story was originally published on the Badger Chemist News website.