Biomolecular condensates provide a way to compartmentalize subcellular components with high temporal and spatial resolution, enabling rapid responses to signals and environmental changes. While the formation, components, and function of some condensates are well-characterized, their presence across organisms, their evolutionary history, and their origin are less well-understood. Here, we review the diversity of condensate components and highlight that not only disordered but also fully structured proteins are capable of driving condensate formation. We compare how proteomes of condensates overlap within and across species, and we present functionally analogous condensates across organisms. Additionally, we discuss the potential role of condensation in early life, suggesting that phase separation could have facilitated the selection and concentration of prebiotic molecules, promoting essential biochemical processes. We conclude that condensate-related organization principles are ubiquitously used across organisms from bacteria to mammals, and they potentially played a key role in prebiotic evolution, serving as primitive compartments for early biochemical processes.

Stop codon readthrough events give rise to longer proteins, which may alter the protein’s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough 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 readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Errors in DNA replication generate genetic mutations, while errors in transcription and translation lead to phenotypic mutations. Phenotypic mutations are orders of magnitude more frequent than genetic ones, yet they are less understood. Here, we review the types of phenotypic mutations, their quantifications, and their role in protein evolution and disease. The diversity generated by phenotypic mutation can facilitate adaptive evolution. Indeed, phenotypic mutations, such as ribosomal frameshift and stop codon readthrough, sometimes serve to regulate protein expression and function. Phenotypic mutations have often been linked to fitness decrease and diseases. Thus, understanding the protein heterogeneity and phenotypic diversity caused by phenotypic mutations will advance our understanding of protein evolution and have implications on human health and diseases.

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 evolutionarily 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.

The complexity of modern proteins makes the understanding of how proteins evolved from simple beginnings a daunting challenge. The Walker-A motif is a phosphate-binding loop (P-loop) found in possibly the most ancient and abundant protein class, so-called P-loop NTPases. By combining phylogenetic analysis and computational protein design, we have generated simple proteins, of only 55 residues, that contain the P-loop and thereby confer binding of a range of phosphate-containing ligands—and even more avidly, RNA and single-strand DNA. Our results show that biochemical function can be implemented in small and simple proteins; they intriguingly suggest that the P-loop emerged as a polynucleotide binder and catalysis of phosphoryl transfer evolved later upon acquisition of higher sequence and structural complexity.

The linkage between regulatory elements of transcription, such as promoters, and their protein products is central to gene function. Promoter–protein coevolution is therefore expected, but rarely observed, and the manner by which these two regulatory levels are linked remains largely unknown. We study glutamate dehydrogenase—a hub of carbon and nitrogen metabolism. In Bacillus subtilis, two paralogues exist: GudB is constitutively transcribed whereas RocG is tightly regulated. In their active, oligomeric states, both enzymes show similar enzymatic rates. However, swaps of enzymes and promoters cause severe fitness losses, thus indicating promoter–enzyme coevolution. Characterization of the proteins shows that, compared to RocG, GudB’s enzymatic activity is highly dependent on glutamate and pH. Promoter–enzyme swaps therefore result in excessive glutamate degradation when expressing a constitutive enzyme under a constitutive promoter, or insufficient activity when both the enzyme and its promoter are tightly regulated. Coevolution of transcriptional and enzymatic regulation therefore underlies paralogue-specific spatio-temporal control, especially under diverse growth conditions.

Margaret Dayhoff’s 1966 hypothesis on the origin of proteins is now an accepted model for the emergence of large, globular, functional proteins from short, simple peptides. However, the fundamental question of how the first protein(s) emerged still stands. The tools and hypotheses pioneered by Dayhoff, and the over 65 million protein sequences and 12 000 structures known today, enable those who follow in her footsteps to address this question.

Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such ‘reconstructed ancestral hyperstability’ can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach. 

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side- chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of ¹HN, ¹⁵N, ¹Ha, ¹³Ca, ¹³CO and ¹³Cb chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.

Simultaneous determination of alcohols, amines, amino acids, flavonoids, and purine and pyrimidine bases in bottled beer samples directly without any pre-treatment was carried out by capillary zone electrophoresis with diode-array detection. Electrolyte conditions such as pH, composition and concentration of the buffer, working voltage and type and time of injection were checked. The best separation of the cited analytes was achieved in 70 mM sodium borate solution and pH 10.25. The detection limits were from 2.1 to 5.6 mg L⁻¹ for the 18 compounds studied. The developed method is rapid, sensitive and quantitative and has been applied to seven types of international bottled beers of different origins bought locally.