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1.  Coevolution analyses illuminate the dependencies between amino acid sites in the chaperonin system GroES-L 
GroESL is a heat-shock protein ubiquitous in bacteria and eukaryotic organelles. This evolutionarily conserved protein is involved in the folding of a wide variety of other proteins in the cytosol, being essential to the cell. The folding activity proceeds through strong conformational changes mediated by the co-chaperonin GroES and ATP. Functions alternative to folding have been previously described for GroEL in different bacterial groups, supporting enormous functional and structural plasticity for this molecule and the existence of a hidden combinatorial code in the protein sequence enabling such functions. Describing this plasticity can shed light on the functional diversity of GroEL. We hypothesize that different overlapping sets of amino acids coevolve within GroEL, GroES and between both these proteins. Shifts in these coevolutionary relationships may inevitably lead to evolution of alternative functions.
We conducted the first coevolution analyses in an extensive bacterial phylogeny, revealing complex networks of evolutionary dependencies between residues in GroESL. These networks differed among bacterial groups and involved amino acid sites with functional importance and others with previously unsuspected functional potential. Coevolutionary networks formed statistically independent units among bacterial groups and map to structurally continuous regions in the protein, suggesting their functional link. Sites involved in coevolution fell within narrow structural regions, supporting dynamic combinatorial functional links involving similar protein domains. Moreover, coevolving sites within a bacterial group mapped to regions previously identified as involved in folding-unrelated functions, and thus, coevolution may mediate alternative functions.
Our results highlight the evolutionary plasticity of GroEL across the entire bacterial phylogeny. Evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins.
PMCID: PMC3728108  PMID: 23875653
2.  Describing the structural robustness landscape of bacterial small RNAs 
The potential role of RNA molecules as gene expression regulators has led to a new perspective on the intracellular control and genome organization. Because secondary structures are crucial for their regulatory role, we sought to investigate their robustness to mutations and environmental changes.
Here, we dissected the structural robustness landscape of the small non-coding RNAs (sncRNAs) encoded in the genome of the bacterium Escherichia coli. We found that bacterial sncRNAs are not significantly robust to both mutational and environmental perturbations when compared against artificial, unbiased sequences. However, we found that, on average, bacterial sncRNAs tend to be significantly plastic, and that mutational and environmental robustness strongly correlate. We further found that, on average, epistasis in bacterial sncRNAs is significantly antagonistic, and positively correlates with plasticity. Moreover, the evolution of robustness is likely dependent upon the environmental stability of the cell, with more fluctuating environments leading to the emergence and fixation of more robust molecules. Mutational robustness also appears to be correlated with structural functionality and complexity.
Our study provides a deep characterization of the structural robustness landscape of bacterial sncRNAs, suggesting that evolvability could be evolved as a consequence of selection for more plastic molecules. It also supports that environmental fluctuations could promote mutational robustness. As a result, plasticity emerges to link robustness, functionality and evolvability.
PMCID: PMC3368786  PMID: 22500888
Evolution; Evolvability; Plasticity; RNA structure; Robustness; Small RNA; Thermodynamics
3.  Relationships of gag-pol diversity between Ty3/Gypsy and Retroviridae LTR retroelements and the three kings hypothesis 
The origin of vertebrate retroviruses (Retroviridae) is yet to be thoroughly investigated, but due to their similarity and identical gag-pol (and env) genome structure, it is accepted that they evolve from Ty3/Gypsy LTR retroelements the retrotransposons and retroviruses of plants, fungi and animals. These 2 groups of LTR retroelements code for 3 proteins rarely studied due to the high variability – gag polyprotein, protease and GPY/F module. In relation to 3 previously proposed Retroviridae classes I, II and II, investigation of the above proteins conclusively uncovers important insights regarding the ancient history of Ty3/Gypsy and Retroviridae LTR retroelements.
We performed a comprehensive study of 120 non-redundant Ty3/Gypsy and Retroviridae LTR retroelements. Phylogenetic reconstruction inferred based on the concatenated analysis of the gag and pol polyproteins shows a robust phylogenetic signal regarding the clustering of OTUs. Evaluation of gag and pol polyproteins separately yields discordant information. While pol signal supports the traditional perspective (2 monophyletic groups), gag polyprotein describes an alternative scenario where each Retroviridae class can be distantly related with one or more Ty3/Gypsy lineages. We investigated more in depth this evidence through comparative analyses performed based on the gag polyprotein, the protease and the GPY/F module. Our results indicate that contrary to the traditional monophyletic view of the origin of vertebrate retroviruses, the Retroviridae class I is a molecular fossil, preserving features that were probably predominant among Ty3/Gypsy ancestors predating the split of plants, fungi and animals. In contrast, classes II and III maintain other phenotypes that emerged more recently during Ty3/Gypsy evolution.
The 3 Retroviridae classes I, II and III exhibit phenotypic differences that delineate a network never before reported between Ty3/Gypsy and Retroviridae LTR retroelements. This new scenario reveals how the diversity of vertebrate retroviruses is polyphyletically recurrent into the Ty3/Gypsy evolution, i.e. older than previously thought. The simplest hypothesis to explain this finding is that classes I, II and III trace back to at least 3 Ty3/Gypsy ancestors that emerged at different evolutionary times prior to protostomes-deuterostomes divergence. We have called this "the three kings hypothesis" concerning the origin of vertebrate retroviruses.
PMCID: PMC2577118  PMID: 18842133
4.  Reducing the false positive rate in the non-parametric analysis of molecular coevolution 
The strength of selective constraints operating on amino acid sites of proteins has a multifactorial nature. In fact, amino acid sites within proteins coevolve due to their functional and/or structural relationships. Different methods have been developed that attempt to account for the evolutionary dependencies between amino acid sites. Researchers have invested a significant effort to increase the sensitivity of such methods. However, the difficulty in disentangling functional co-dependencies from historical covariation has fuelled the scepticism over their power to detect biologically meaningful results. In addition, the biological parameters connecting linear sequence evolution to structure evolution remain elusive. For these reasons, most of the evolutionary studies aimed at identifying functional dependencies among protein domains have focused on the structural properties of proteins rather than on the information extracted from linear multiple sequence alignments (MSA). Non-parametric methods to detect coevolution have been reported to be especially susceptible to produce false positive results based on the properties of MSAs. However, no formal statistical analysis has been performed to definitively test the differential effects of these properties on the sensitivity of such methods.
Here we test the effect that variations on the MSA properties have over the sensitivity of non-parametric methods to detect coevolution. We test the effect that the size of the MSA (number of sequences), mean pairwise amino acid distance per site and the strength of the coevolution signal have on the ability of non-parametric methods to detect coevolution. Our results indicate that all three factors have significant effects on the accuracy of non-parametric methods. Further, introducing statistical filters improves the sensitivity and increases the statistical power of the methods to detect functional coevolution. Statistical analysis of the physico-chemical properties of amino acid sites in the context of the protein structure reveals striking dependencies among amino acid sites. Results indicate a covariation trend in the hydrophobicities and molecular weight characteristics of amino acid sites when analysing a non-redundant set of 8000 protein structures. Using this biological information as filter in coevolutionary analyses minimises the false positive rate of these methods. Application of these filters to three different proteins with known functional domains supports the importance of using biological filters to detect coevolution.
Coevolutionary analyses using non-parametric methods have proved difficult and highly prone to provide spurious results depending on the properties of MSAs and on the strength of coevolution between amino acid sites. The application of statistical filters to the number of pairs detected as coevolving reduces significantly the number of artifactual results. Analysis of the physico-chemical properties of amino acid sites in the protein structure context reveals their structure-dependent covariation. The application of this known biological information to the analysis of covariation greatly enhances the functional coevolutionary signal and removes historical covariation. Simultaneous use of statistical and biological data is instrumental in the detection of functional amino acid sites dependencies and compensatory changes at the protein level.
PMCID: PMC2362121  PMID: 18402697
5.  Mutational dynamics of murine angiogenin duplicates 
Angiogenin (Ang) is a protein involved in angiogenesis by inducing the formation of blood vessels. The biomedical importance of this protein has come from findings linking mutations in Ang to cancer progression and neurodegenerative diseases. These findings highlight the evolutionary constrain on Ang amino acid sequence. However, previous studies comparing human Angiogenin with homologs from other phylogenetically related organisms have led to the conclusion that Ang presents a striking variability. Whether this variability has an adaptive value per se remains elusive. Understanding why many functional Ang paralogs have been preserved in mouse and rat and identifying functional divergence mutations at these copies may explain the relationship between mutations and function. In spite of the importance of testing this hypothesis from the evolutionarily and biomedical perspectives, this remains yet unaccomplished. Here we test the main mutational dynamics driving the evolution and function of Ang paralogs in mammals.
We analysed the phylogenetic asymmetries between the different Ang gene copies in mouse and rat in the context of vertebrate Ang phylogeny. This analysis shows strong evidence in support of accelerated evolution in some Ang murine copies (mAng). This acceleration is not due to non-functionalisation because constraints on amino acid replacements remain strong. We identify many of the amino acid sites involved in signal localization and nucleotide binding by Ang to have evolved under diversifying selection. Compensatory effects of many of the mutations at these paralogs and their key structural location in or nearby important functional regions support a possible functional shift (functional divergence) in many Ang copies. Similarities between 3D-structural models for mAng copies suggest that their divergence is mainly functional.
We identify the main evolutionary dynamics shaping the variability of Angiogenin in vertebrates and highlight the plasticity of this protein after gene duplication. Our results suggest functional divergence among mAng paralogs. This puts forward mAng as a good system candidate for testing functional plasticity of such an important protein while stresses caution when using mouse as a model to infer the consequences of mutations in the single Ang copy of humans.
PMCID: PMC2964713  PMID: 20950426
6.  In silico identification of functional divergence between the multiple groEL gene paralogs in Chlamydiae 
Heat-shock proteins are specialized molecules performing different and essential roles in the cell including protein degradation, folding and trafficking. GroEL is a 60 Kda heat-shock protein ubiquitous in bacteria and has been regarded as an important molecule implicated in chronic inflammatory processes caused by Chlamydiae infections. GroEL in Chlamydiae became duplicated at the origin of the Chlamydiae lineage presenting three distinct molecular chaperones, namely the original protein GroEL1 (Ct110), and its paralogous proteins GroEL2 (Ct604) and GroEL3 (Ct755). These chaperones present differential and independent expressions during the different stages of Chlamydiae infections and have been suggested to present differential physiological and regulatory roles.
In this comprehensive in silico study we show that GroEL protein paralogs have diverged functionally after the different gene duplication events and that this divergence has occurred mainly between GroEL3 and GroEL1. GroEL2 presents an intermediate functional divergence pattern from GroEL1. Our results point to the different protein-protein interaction patterns between GroEL paralogs and known GroEL protein clients supporting their functional divergence after groEL gene duplication. Analysis of selective constraints identifies periods of adaptive evolution after gene duplication that led to the fixation of amino acid replacements in GroEL protein domains involved in the interaction with GroEL protein clients.
We demonstrate that GroEL protein copies in Chlamydiae species have diverged functionally after the gene duplication events. We also show that functional divergence has occurred in important functional regions of these GroEL proteins and that very probably have affected the ancestral GroEL regulatory role and protein-protein interaction patterns with GroEL client proteins. Most of the amino acid replacements that have affected interaction with protein clients and that were responsible for the functional divergence between GroEL paralogs were fixed by adaptive evolution after the groEL gene duplication events.
PMCID: PMC1892554  PMID: 17519003

Results 1-6 (6)