Biology subjects

Romero-Romero, M. L.

Publications and source records attributed to Romero-Romero, M. L..

3 recordsLinked to original sources

bioRxiv2023

ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON MISCODING

Stop codon miscoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon miscoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon miscoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon miscoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon miscoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon miscoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon miscoding in E.coli. Overall, our findings suggest that the environment influences the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

systems biology↗

bioRxiv2022

Active site center redesign increases protein stability preserving catalysis

The stabilization of natural proteins is a long-standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged-to-hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by Partial Least-squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stability reported for a protein with a natural occurring fold (138 {degrees}C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo. SIGNIFICANCEThe major driving force of protein folding is the hydrophobic effect, and increasing the protein core hydrophobicity essentially increases protein stability. Active sites often contain buried ionizable groups, which can be essential for function but dramatically reduce protein stability. Thus, increasing the protein core hydrophobicity cannot be applied to enzyme active sites without a functional cost. We propose a method to enhance protein stability by overcoming this obstacle. We show that catalytic properties of buried charges can be mimicked with surface mutations, thus paving the way to unlock the optimization of the hydrophobic core to stabilize enzymes.

biophysics↗

bioRxiv2019

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionary conserved, but span at least a [~]100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding-assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

biophysics↗