Development of an unbiased statistical method for the analysis of unigenic evolution

Background  

Unigenic evolution is a powerful genetic strategy involving random mutagenesis of a single gene product to delineate functionally important domains of a protein. This method involves selection of variants of the protein which retain function, followed by statistical analysis comparing expected and observed mutation frequencies of each residue. Resultant mutability indices for each residue are averaged across a specified window of codons to identify hypomutable regions of the protein. As originally described, the effect of changes to the length of this averaging window was not fully eludicated. In addition, it was unclear when sufficient functional variants had been examined to conclude that residues conserved in all variants have important functional roles.     

Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution

Pin1 is a phosphorylation-dependent member of the parvulin family of peptidyl-prolyl isomerases exhibiting functional conservation between yeast and man. To perform an unbiased analysis of the regions of Pin1 essential for its functions, we generated libraries of randomly mutated forms of the human Pin1 cDNA and identified functional Pin1 alleles by their ability to complement the Pin1 homolog Ess1 in Saccharomyces cerevisiae. We isolated an extensive collection of functional mutant Pin1 clones harboring a total of 356 amino acid substitutions. Surprisingly, many residues previously thought to be critical in Pin1 were found to be altered in this collection of functional mutants. In fact, only 17 residues were completely conserved in these mutants and in Pin1 sequences from other eukaryotic organisms, with only two of these conserved residues located within the WW domain of Pin1. Examination of invariant residues provided new insights regarding a phosphate-binding loop that distinguishes a phosphorylation-dependent peptidyl-prolyl isomerase such as Pin1 from other parvulins. In addition, these studies led to an investigation of residues involved in catalysis including C113 that was previously implicated as the catalytic nucleophile. We demonstrate that substitution of C113 with D does not compromise Pin1 function in vivo nor does this substitution abolish catalytic activity in purified recombinant Pin1. These findings are consistent with the prospect that the function of residue 113 may not be that of a nucleophile, thus raising questions about the model of nucleophilic catalysis. Accordingly, an alternative catalytic mechanism for Pin1 is postulated.     

Estimating the evidence of selection and the reliability of inference in unigenic evolution

Background  

Unigenic evolution is a large-scale mutagenesis experiment used to identify residues that are potentially important for protein function. Both currently-used methods for the analysis of unigenic evolution data analyze 'windows' of contiguous sites, a strategy that increases statistical power but incorrectly assumes that functionally-critical sites are contiguous. In addition, both methods require the questionable assumption of asymptotically-large sample size due to the presumption of approximate normality.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Science China Life Sciences - Naturally occurring structural variations (SVs) are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes. Controllable...     

Fluctuation domains in adaptive evolution

We derive an expression for the variation between parallel trajectories in phenotypic evolution, extending the well known result that predicts the mean evolutionary path in adaptive dynamics or quantitative genetics. We show how this expression gives rise to the notion of fluctuation domains-parts of the fitness landscape where the rate of evolution is very predictable (due to fluctuation dissipation) and parts where it is highly variable (due to fluctuation enhancement). These fluctuation domains are determined by the curvature of the fitness landscape. Regions of the fitness landscape with positive curvature, such as adaptive valleys or branching points, experience enhancement. Regions with negative curvature, such as adaptive peaks, experience dissipation. We explore these dynamics in the ecological scenarios of implicit and explicit competition for a limiting resource.     

Experimental evolution of a sexually selected display in yeast

The fundamental principle underlying sexual selection theory is that an allele conferring an advantage in the competition for mates will spread through a population. Remarkably, this has never been demonstrated empirically. We have developed an experimental system using yeast for testing genetic models of sexual selection. Yeast signal to potential partners by producing an attractive pheromone; stronger signallers are preferred as mates. We tested the effect of high and low levels of sexual selection on the evolution of a gene determining the strength of this signal. Under high sexual selection, an allele encoding a stronger signal was able to invade a population of weak signallers, and we observed a corresponding increase in the amount of pheromone produced. By contrast, the strong signalling allele failed to invade under low sexual selection. Our results demonstrate, for the first time, the spread of a sexually selected allele through a population, confirming the central assumption of sexual selection theory. Our yeast system is a powerful tool for investigating the genetics of sexual selection.     

Protein evolution within a structural space

Understanding of the evolutionary origins of protein structures represents a key component of the understanding of molecular evolution as a whole. Here we seek to elucidate how the features of an underlying protein structural “space” might impact protein structural evolution. We approach this question using lattice polymers as a completely characterized model of this space. We develop a measure of structural comparison of lattice structures that is analogous to the one used to understand structural similarities between real proteins. We use this measure of structural relatedness to create a graph of lattice structures and compare this graph (in which nodes are lattice structures and edges are defined using structural similarity) to the graph obtained for real protein structures. We find that the graph obtained from all compact lattice structures exhibits a distribution of structural neighbors per node consistent with a random graph. We also find that subgraphs of 3500 nodes chosen either at random or according to physical constraints also represent random graphs. We develop a divergent evolution model based on the lattice space which produces graphs that, within certain parameter regimes, recapitulate the scale-free behavior observed in similar graphs of real protein structures.     

Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis

The two domains of yeast phosphoglycerate kinase were produced by recombinant techniques. The N-domain was obtained by the introduction of a termination codon at the position coding for Phe185, and the C-domain by a deletion in the gene of the coding sequence between Ser1 and Leu186. Both domains were efficiently expressed in yeast, the level for the C-domain being greater than that for the N-domain. Both domains were found to have a quasi-native structure; the C-domain retained its ability to bind nucleotides. Small local differences were detected in domain structure compared to that in the whole enzyme, probably due to the lack of interdomain stabilizing interactions. Nevertheless, such an approach provides direct evidence for independent folding of domains in a two-domain protein.     

Rapid divergence in the course of Drosophila evolution reveals structural important domains of the Notch antagonist Hairless

 Hairless is a member of the Notch signalling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless orthologue from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far probably due to a low degree of conservation. Therefore, we turned to D. hydei where a Hairless mutation has been described before. The D. hydei Hairless orthologue is reasonably well conserved with regard to gene structure and expression. The prospective Hairless protein orthologues share several highly conserved regions which are separated by quite diverged stretches. As to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support our earlier structure-function analysis since they nicely correspond to previously defined, functionally important protein domains. Most notably, the very C-terminal domain which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologues in other animals, especially in vertebrates. Received: 26 August 1998 / Accepted: 9 November 1998     

3Dee: a database of protein structural domains

The 3Dee database is a repository of protein structural domains. It stores alternative domain definitions for the same protein, organises domains into sequence and structural hierarchies, contains non-redundant set(s) of sequences and structures, multiple structure alignments for families of domains, and allows previous versions of the database to be regenerated. AVAILABILITY: 3Dee is accessible on the World Wide Web at the URL http://barton.ebi.ac.uk/servers/3Dee.html.     

Energetics of structural domains in alpha-lactalbumin.

alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.     

Functional and structural analyses of mouse genomic regions screened by the morphological specific-locus test

Genetic analyses of certain classes of mutations recovered in the mouse specific-locus test (SLT) have characterized arrays of deletions, overlapping at the marked loci. Complementation maps, generated for several of the regions, have identified a number of functional units surrounding each marked locus and have ordered the mutations into complementation groups. Molecular entry to all but one of the marked regions has been achieved by (1) identifying proviral integrations in, or close to, the specific loci (d, se, a, c); (2) mapping random anonymous clones from appropriately enriched libraries to the longest deleted segments, then submapping to more limited segments on the basis of complementation and deletion-breakpoint maps (c, p); (3) similarly mapping known clones thought to be located in pertinent chromosomal regions (p, c, d); and (4) cloning specific genes that reside in regions corresponding to the deletions (b, c, p). The molecular analyses have confirmed that genetically-inferred deletions are structural deletions of DNA. The emerging physical maps are concordant with the complementation maps, and in several cases have discriminated among members of a complementation group with respect to breakpoint positions. Deletion-breakpoint-fusion fragments have prove to be highly useful for making large chromosomal jumps to facilitate physical mapping. The recent advances toward correlating physical and functional maps of specific regions of the mouse genome owe much to the existence of arrays of mutations involving loci marked in the SLT. In turn, the characterizations of these regions have made it possible to demonstrate qualitative differences among mutations resulting from different treatments. This new capability for qualitative analysis, which will increase as the molecular studies proceed, further enhances the value of the SLT, which has been extensively used for quantitative studies in germ-cell mutagenesis.     

Identification of structural domains in proteins by a graph heuristic.

A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.