Rather than poach proteins singly, scientists at ETH Zurich turned up the heat on entire proteomes. Some proteins proved to be especially sensitive to heat, collapsing or denaturing sooner than most of their peers. Even though the most sensitive proteins represent a minority, they are, apparently, crucial to cell survival. Without these proteins, cells perish even when most of their proteins remain intact, despite simmering conditions.
These findings have several implications. To draw them out, the ETH Zurich scientists considered how structural and functional variations among proteins correlated with heat sensitivity. Ultimately, the scientists offered their take on the molecular and evolutionary bases of protein and proteome stability. In general, the scientists emphasized that cells live with a trade-off between protein flexibility and protein stability. While flexible proteins are better able to carry out varying tasks inside a cell, they tend to be more unstable—and more vulnerable to heat.
Details appeared February 24 in the journal Science, in an article entitled, “Cell-Wide Analysis of Protein Thermal Unfolding Reveals Determinants of Thermostability.” It describes how the ETH scientists studied the heat stability of proteins more systematically than previous studies, which tended to rely on computational models, measurements obtained using protein-distorting labels, or observations of individual proteins in dilute solutions.
The ETH scientists preserved the cellular milieu and considered the full range of proteins by applying a structural proteome approach. They combined limited proteolysis and mass spectrometry, and they expanded their evaluations over a range of temperatures and across three live organisms—Escherichia coli, Saccharomyces cerevisiae, and Thermus thermophilus—as well as human cells. Eventually, the scientists compiled thermostability data for more than 8000 proteins.
The authors of the Science article summarized their work as follows: “Our results (i) indicate that temperature-induced cellular collapse is due to the loss of a subset of proteins with key functions, (ii) shed light on the evolutionary conservation of protein and domain stability, and (iii) suggest that natively disordered proteins in a cell are less prevalent than predicted and (iv) that highly expressed proteins are stable because they are designed to tolerate translational errors that would lead to the accumulation of toxic misfolded species.”
Previous research based on computational analysis has assumed that a large part of the proteins of a cell denature when the narrow temperature range in which the proteins function optimally is exceeded. For the intestinal bacterium E. coli, the optimal temperature is about 37°C; anything above 46°C and the bacteria die because the protein structures collapse.
“Thanks to this research, we can now show that only a few proteins collapse at the temperature at which the bacterium dies,” said Paola Picotti, Ph.D., assistant professor of biochemistry at ETH Zurich. “We could not confirm the prediction that the majority of proteins of an organism denature at the same time.”
About 80 of the proteins examined collapsed as soon as the temperature exceeded the species-specific optimum by a few degrees. Although they constitute only a small fraction of the proteins of a cell, this proves fatal for the cell since some of these types of proteins have vital functions or are key components in a large protein network. “As soon as these key components fail, the cell cannot continue to function,” noted Dr. Picotti.
That the key components of a biological system are sensitive to heat would at first glance appear to be an evolutionary glitch. However, these proteins are often unstable as a result of their flexibility, which enables them to carry out varying tasks in the cell, says the biochemist. “Flexibility and stability can be mutually exclusive. The cell has to make a compromise.”
The researchers also show that the most stable proteins and the least prone to aberrant or pathological folds are also the most common in cells. From the perspective of the cell, this makes the most sense. Were it reversed and the most common proteins were to misfold the fastest, the cell would have to invest a lot of energy in their reconstruction or disposal. For this reason, cells ensure that common proteins are more stable than the rare ones.
But why are T. thermophilus bacteria unaffected even by temperatures of over 70°C? According to the researchers, these cells would preferentially stabilize the more heat-sensitive, functionally crucial proteins, such as through adapted protein sequences.
The findings of the current study could be used to help genetically modify organisms to withstand higher temperatures. Today, certain chemicals, such as ethanol, are biotechnologically produced with the help of bacteria. But these bacteria often work only in a narrow temperature window, which constrains the yield. If production could proceed at higher temperatures, the yield could be optimized without damage to the bacteria.
The researchers also found evidence that certain denatured proteins tend to clump again at even higher temperatures and form aggregates. In human cells, Dr. Picotti and her colleagues found that the protein DNMT1 first denatures with increasing heat and later aggregates with others of its kind. These and other proteins with similar properties are associated with neurological disorders, such as Alzheimer's or Parkinson's.
This study is the first to investigate the thermal stability of proteins from several organisms on a large scale directly in the complex cellular matrix. Proteins were neither isolated from the cellular fluid nor purified to conduct the measurements. For their study, the researchers broke the cells open and then measured the stability of all proteins directly in the cellular fluid at different temperatures.