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How proteomes take the heat
Living organisms are very sensitive to temperature, and much of this is attributed to its effect on the structure and function of proteins. Leuenberger et al. explored thermostability on a proteome-wide scale in bacteria, yeast, and human cells by using a combination of limited proteolysis and mass spectrometry (see the Perspective by Vogel). Their results suggest that temperature-induced cell death is caused by the loss of a subset of proteins with key functions. The study also provides insight into the molecular and evolutionary bases of protein and proteome stability.
Structured Abstract
INTRODUCTION
Temperature is crucially important to life. Small temperature changes can differentiate optimal and lethal growth conditions of living organisms. Because of the higher abundance and lower stability of proteins as compared with those of other biological macromolecules, thermally induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized.
RATIONALE
To determine the thermal stability of proteins on a proteome-wide scale and with domain-level resolution, we developed a structural proteomic approach that relies on limited proteolysis (LiP) and mass spectrometry (MS) applied over a range of temperatures.
RESULTS
Our LiP-MS strategy was validated through analysis of purified proteins in the presence and absence of a biologically relevant matrix. We then obtained proteome-wide thermal denaturation profiles for Escherichia coli, Saccharomyces cerevisiae, Thermus thermophilus, and human cells. In contrast to previous predications that proteome instability derives from the simultaneous and generalized loss of hundreds of proteins, we observed that at a temperature at which cells experience temperature-induced physiological impairment, a subset of essential proteins undergoes denaturation.
Confirming results of previous studies on the basis of comparison of genomes of thermophilic and mesophilic bacteria, we observed enrichment for lysine residues and β-sheet structures in thermostable proteins. We also found that unstable proteins have a higher content of aspartic acid than that of stable proteins and observed an inverse correlation between protein length and thermal stability. Further, thermostable proteins are substantially less prone to thermal aggregation than unstable proteins.
Relative domain thermostability was conserved both within species and across organisms. Thermal stability was not generally similar for proteins encoded by orthologous genes. This suggests that the melting temperatures of proteins are affected by the reshuffling of protein domains, despite the conservation of domain stability.
According to the “translational robustness” theory, highly expressed proteins must tolerate translational errors that can lead to the accumulation of toxic misfolded species. Our data show a clear direct relationship between protein thermal stability and intracellular abundance and an inverse relationship between protein stability and aggregation or local unfolding. Increasing the thermodynamic stabilities of the folds of abundant proteins will broaden the range of amino acid replacements that a protein can tolerate before misfolding. Our findings suggest that over the course of evolution, the burden of intracellular misfolding has been reduced by increasing the thermodynamic stability of abundant proteins. Last, although up to 30% of proteomes have been predicted to consist of intrinsically disordered proteins (IDPs), our data revealed that about half of these proteins showed two-state denaturation profiles in the cellular matrix. This suggests that many IDPs are globally or locally structured in cells.
CONCLUSION
Our study contributes insight into the molecular and evolutionary bases of protein and proteome thermostability and provides a blueprint for future studies on the stability of proteomes and thermal denaturation.
Node color indicates protein thermostability. Blue, unstable; yellow, medium-stable; orange, stable; gray, not measured. At the temperature of thermal cell death of E. coli, a subset of highly connected protein nodes involved in key cellular processes undergoes temperature-induced denaturation.
Abstract
Temperature-induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized. We developed a structural proteomic strategy to measure protein thermostability on a proteome-wide scale and with domain-level resolution. We applied it to Escherichia coli, Saccharomyces cerevisiae, Thermus thermophilus, and human cells, yielding thermostability data for more than 8000 proteins. 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.
