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Abstract Background The number of gene sequences that are available for comparative genomics approaches is increasing extremely quickly. A current challenge is to be able to handle this huge amount of sequences in order to build families of homologous sequences in a reasonable time. Results We present the software package SiLiX that implements a novel method which reconsiders single linkage clustering with a graph theoretical approach. A parallel version of the algorithms is also presented. As a demonstration of the ability of our software, we clustered more than 3 millions sequences from about 2 billion BLAST hits in 7 minutes, with a high clustering quality, both in terms of sensitivity and specificity. Conclusions Comparing state-of-the-art software, SiLiX presents the best up-to-date capabilities to face the problem of clustering large collections of sequences. SiLiX is freely available at http://lbbe.univ-lyon1.fr/SiLiX.

In phylogenomics, incongruences between gene trees, resulting from both artifactual and biological reasons, can decrease the signal-to-noise ratio and complicate species tree inference. The amount of data handled today in classical phylogenomic analyses precludes manual error detection and removal. However, a simple and efficient way to automate the identification of outliers from a collection of gene trees is still missing. Here, we present PhylteR, a method that allows rapid and accurate detection of outlier sequences in phylogenomic datasets, i.e. species from individual gene trees that do not follow the general trend. PhylteR relies on DISTATIS, an extension of multidimensional scaling to 3 dimensions to compare multiple distance matrices at once. In PhylteR, these distance matrices extracted from individual gene phylogenies represent evolutionary distances between species according to each gene. On simulated datasets, we show that PhylteR identifies outliers with more sensitivity and precision than a comparable existing method. We also show that PhylteR is not sensitive to ILS-induced incongruences, which is a desirable feature. On a biological dataset of 14,463 genes for 53 species previously assembled for Carnivora phylogenomics, we show (i) that PhylteR identifies as outliers sequences that can be considered as such by other means, and (ii) that the removal of these sequences improves the concordance between the gene trees and the species tree. Thanks to the generation of numerous graphical outputs, PhylteR also allows for the rapid and easy visual characterization of the dataset at hand, thus aiding in the precise identification of errors. PhylteR is distributed as an R package on CRAN and as containerized versions (docker and singularity)., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Many layouts exist for visualizing phylogenetic trees, allowing to display the same information (evolutionary relationships) in different ways. For large phylogenies, the choice of the layout is a key element, because the printable area is limited, and because interactive on-screen visualizers can lead to unreadable phylogenetic relationships at high zoom levels. A visual inspection of available layouts for rooted trees reveals large empty areas that one may want to fill in order to use less drawing space and eventually gain readability. This can be achieved by using the nonlayered tidy tree layout algorithm that was proposed earlier but was never used in a phylogenetic context so far. Here, we present its implementation, and we demonstrate its advantages on simulated and biological data (the measles virus phylogeny). Our results call for the integration of this new layout in phylogenetic software. We implemented the nonlayered tidy tree layout in R language as a stand-alone function (available at https://github.com/damiendevienne/non-layered-tidy-trees), as an option in the tree plotting function of the R package ape, and in the recent tool for visualizing reconciled phylogenetic trees thirdkind (https://github.com/simonpenel/thirdkind/wiki)., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Motivation: Reconciliation between a host and its symbiont phylogenies or between a species and a gene phylogenies is a prevalent approach in evolution, however no simple generic tool (i.e. virtually usable by all reconciliation software, from host/symbiont to species/gene comparisons) is available to visualize reconciliation results. Moreover there is no tool to visualize 3-levels reconciliations, i.e. to visualize 2 nested reconciliations as for example in a host/symbiont/gene complex., Results: Thirdkind is a light and easy to install command line software producing svg files displaying reconciliations, including 3-levels reconciliations. It takes a standard format recPhyloXML as input, and is thus usable with most reconciliation software., Availability and Implementation: https://github.com/simonpenel/thirdkind/wiki., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Paramecium aurelia complex, a group of morphologically similar but sexually incompatible sibling species, is a unique example of the evolutionary plasticity of mating-type systems. Each species has two mating types, O (Odd) and E (Even). Although O and E types are homologous in all species, three different modes of determination and inheritance have been described: genetic determination by Mendelian alleles, stochastic developmental determination, and maternally inherited developmental determination. Previous work in three species of the latter kind has revealed the key roles of the E-specific transmembrane protein mtA and its highly specific transcription factor mtB: type O clones are produced by maternally inherited genome rearrangements that inactivate either mtA or mtB during development. Here we show, through transcriptome analyses in five additional species representing the three determination systems, that mtA expression specifies type E in all cases. We further show that the Mendelian system depends on functional and nonfunctional mtA alleles, and identify novel developmental rearrangements in mtA and mtB which now explain all cases of maternally inherited mating-type determination. Epistasis between these genes likely evolved from less specific interactions between paralogs in the P. aurelia common ancestor, after a whole-genome duplication, but the mtB gene was subsequently lost in three P. aurelia species which appear to have returned to an ancestral regulation mechanism. These results suggest a model accounting for evolutionary transitions between determination systems, and highlight the diversity of molecular solutions explored among sibling species to maintain an essential mating-type polymorphism in cell populations., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Sex chromosomes are typically comprised of a non-recombining region and a recombining pseudoautosomal region. Accurately quantifying the relative size of these regions is critical for sex-chromosome biology both from a functional and evolutionary perspective. The evolution of the pseudoautosomal boundary (PAB) is well documented in haplorrhines (apes and monkeys) but not in strepsirrhines (lemurs and lorises). Here, we studied the PAB of seven species representing the main strepsirrhine lineages by sequencing a male and a female genome in each species and using sex differences in coverage to identify the PAB. We found that during primate evolution, the PAB has remained unchanged in strepsirrhines whereas several recombination suppression events moved the PAB and shortened the pseudoautosomal region in haplorrhines. Strepsirrhines are well known to have much lower sexual dimorphism than haplorrhines. We suggest that mutations with antagonistic effects between males and females have driven recombination suppression and PAB evolution in haplorrhines., Competing Interests: RS, AW, SM, CR, SP, JL, GD, BC, EL, GM No competing interests declared, (© 2020, Shearn et al.)

Motivation: Gene and species tree reconciliation methods are used to interpret gene trees, root them and correct uncertainties that are due to scarcity of signal in multiple sequence alignments. So far, reconciliation tools have not been integrated in standard phylogenetic software and they either lack performance on certain functions, or usability for biologists., Results: We present Treerecs, a phylogenetic software based on duplication-loss reconciliation. Treerecs is simple to install and to use. It is fast and versatile, has a graphic output, and can be used along with methods for phylogenetic inference on multiple alignments like PLL and Seaview., Availability and Implementation: Treerecs is open-source. Its source code (C++, AGPLv3) and manuals are available from https://project.inria.fr/treerecs/., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Horizontal gene transfer (HGT) is considered as a major source of innovation in bacteria, and as such is expected to drive adaptation to new ecological niches. However, among the many genes acquired through HGT along the diversification history of genomes, only a fraction may have actively contributed to sustained ecological adaptation. We used a phylogenetic approach accounting for the transfer of genes (or groups of genes) to estimate the history of genomes in Agrobacterium biovar 1, a diverse group of soil and plant-dwelling bacterial species. We identified clade-specific blocks of cotransferred genes encoding coherent biochemical pathways that may have contributed to the evolutionary success of key Agrobacterium clades. This pattern of gene coevolution rejects a neutral model of transfer, in which neighboring genes would be transferred independently of their function and rather suggests purifying selection on collectively coded acquired pathways. The acquisition of these synapomorphic blocks of cofunctioning genes probably drove the ecological diversification of Agrobacterium and defined features of ancestral ecological niches, which consistently hint at a strong selective role of host plant rhizospheres., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The field of stoichiogenomics aims at understanding the influence of nutrient limitations on the elemental composition of the genome, transcriptome, and proteome. The 20 amino acids and the 4 nt differ in the number of nutrients they contain, such as nitrogen (N). Thus, N limitation shall theoretically select for changes in the composition of proteins or RNAs through preferential use of N-poor amino acids or nucleotides, which will decrease the N-budget of an organism. While these N-saving mechanisms have been evidenced in microorganisms, they remain controversial in multicellular eukaryotes. In this study, we used 13 surface and subterranean isopod species pairs that face strongly contrasted N limitations, either in terms of quantity or quality. We combined in situ nutrient quantification and transcriptome sequencing to test if N limitation selected for N-savings through changes in the expression and composition of the transcriptome and proteome. No evidence of N-savings was found in the total N-budget of transcriptomes or proteomes or in the average protein N-cost. Nevertheless, subterranean species evolving in N-depleted habitats displayed lower N-usage at their third codon positions. To test if this convergent compositional change was driven by natural selection, we developed a method to detect the strand-asymmetric signature that stoichiogenomic selection should leave in the substitution pattern. No such signature was evidenced, indicating that the observed stoichiogenomic-like patterns were attributable to nonadaptive processes. The absence of stoichiogenomic signal despite strong N limitation within a powerful phylogenetic framework casts doubt on the existence of stoichiogenomic mechanisms in metazoans., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ribosomal proteins (r-proteins) are increasingly used as an alternative to ribosomal rRNA for prokaryotic systematics. However, their routine use is difficult because r-proteins are often not or wrongly annotated in complete genome sequences, and there is currently no dedicated exhaustive database of r-proteins. RiboDB aims at fulfilling this gap. This weekly updated comprehensive database allows the fast and easy retrieval of r-protein sequences from publicly available complete prokaryotic genome sequences. The current version of RiboDB contains 90 r-proteins from 3,750 prokaryotic complete genomes encompassing 38 phyla/major classes and 1,759 different species. RiboDB is accessible at http://ribodb.univ-lyon1.fr and through ACNUC interfaces., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Motivation: Proteins can be naturally classified into families of homologous sequences that derive from a common ancestor. The comparison of homologous sequences and the analysis of their phylogenetic relationships provide useful information regarding the function and evolution of genes. One important difficulty of clustering methods is to distinguish highly divergent homologous sequences from sequences that only share partial homology due to evolution by protein domain rearrangements. Existing clustering methods require parameters that have to be set a priori. Given the variability in the evolution pattern among proteins, these parameters cannot be optimal for all gene families., Results: We propose a strategy that aims at clustering sequences homologous over their entire length, and that takes into account the pattern of substitution specific to each gene family. Sequences are first all compared with each other and clustered into pre-families, based on pairwise similarity criteria, with permissive parameters to optimize sensitivity. Pre-families are then divided into homogeneous clusters, based on the topology of the similarity network. Finally, clusters are progressively merged into families, for which we compute multiple alignments, and we use a model selection technique to find the optimal tradeoff between the number of families and multiple alignment likelihood. To evaluate this method, called HiFiX, we analyzed simulated sequences and manually curated datasets. These tests showed that HiFiX is the only method robust to both sequence divergence and domain rearrangements. HiFiX is fast enough to be used on very large datasets., Availability and Implementation: The Python software HiFiX is freely available at http://lbbe.univ-lyon1.fr/hifix.

Background: The number of gene sequences that are available for comparative genomics approaches is increasing extremely quickly. A current challenge is to be able to handle this huge amount of sequences in order to build families of homologous sequences in a reasonable time., Results: We present the software package SiLiX that implements a novel method which reconsiders single linkage clustering with a graph theoretical approach. A parallel version of the algorithms is also presented. As a demonstration of the ability of our software, we clustered more than 3 millions sequences from about 2 billion BLAST hits in 7 minutes, with a high clustering quality, both in terms of sensitivity and specificity., Conclusions: Comparing state-of-the-art software, SiLiX presents the best up-to-date capabilities to face the problem of clustering large collections of sequences. SiLiX is freely available at http://lbbe.univ-lyon1.fr/SiLiX.

Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus-virus and virus-host lateral gene transfers. PhEVER (http://pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems.

Background: Comparative genomics is a central step in many sequence analysis studies, from gene annotation and the identification of new functional regions in genomes, to the study of evolutionary processes at the molecular level (speciation, single gene or whole genome duplications, etc.) and phylogenetics. In that context, databases providing users high quality homologous families and sequence alignments as well as phylogenetic trees based on state of the art algorithms are becoming indispensable., Methods: We developed an automated procedure allowing massive all-against-all similarity searches, gene clustering, multiple alignments computation, and phylogenetic trees construction and reconciliation. The application of this procedure to a very large set of sequences is possible through parallel computing on a large computer cluster., Results: Three databases were developed using this procedure: HOVERGEN, HOGENOM and HOMOLENS. These databases share the same architecture but differ in their content. HOVERGEN contains sequences from vertebrates, HOGENOM is mainly devoted to completely sequenced microbial organisms, and HOMOLENS is devoted to metazoan genomes from Ensembl. Access to the databases is provided through Web query forms, a general retrieval system and a client-server graphical interface. The later can be used to perform tree-pattern based searches allowing, among other uses, to retrieve sets of orthologous genes. The three databases, as well as the software required to build and query them, can be used or downloaded from the PBIL (Pôle Bioinformatique Lyonnais) site at http://pbil.univ-lyon1.fr/.

A stringent branch-site codon model was used to detect positive selection in vertebrate evolution. We show that the test is robust to the large evolutionary distances involved. Positive selection was detected in 77% of 884 genes studied. Most positive selection concerns a few sites on a single branch of the phylogenetic tree: Between 0.9% and 4.7% of sites are affected by positive selection depending on the branches. No functional category was overrepresented among genes under positive selection. Surprisingly, whole genome duplication had no effect on the prevalence of positive selection, whether the fish-specific genome duplication or the two rounds at the origin of vertebrates. Thus positive selection has not been limited to a few gene classes, or to specific evolutionary events such as duplication, but has been pervasive during vertebrate evolution.

Single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variations in numerous organisms, have emerged as important tools for the study of complex genetic traits and deciphering of genome evolution. High-throughput genome sequencing projects worldwide provide an unprecedented opportunity for whole-genome SNP analysis in a variety of species. To facilitate SNP discovery in vertebrates, we have developed a web-based, user-friendly, and fully automated application, DigiPINS, for genome-wide identification of exonic SNPs from EST data. Currently, the database can be used to the mining of exonic SNPs in six complete genomes (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus and Danio rerio). In addition to providing information on sequence conservation, DigiPINS allows compilation of comprehensive sets of polymorphisms within cancer candidate genes or identification of novel cancer markers, making it potentially useful for cancer association studies. The DigiPINS server is available via the internet at http://pbil.univ-lyon1.fr/gem/DigiPINS/query_DigiPINS.php.

Integr8 is a new web portal for exploring the biology of organisms with completely deciphered genomes. For over 190 species, Integr8 provides access to general information, recent publications, and a detailed statistical overview of the genome and proteome of the organism. The preparation of this analysis is supported through Genome Reviews, a new database of bacterial and archaeal DNA sequences in which annotation has been upgraded (compared to the original submission) through the integration of data from many sources, including the EMBL Nucleotide Sequence Database, the UniProt Knowledgebase, InterPro, CluSTr, GOA and HOGENOM. Integr8 also allows the users to customize their own interactive analysis, and to download both customized and prepared datasets for their own use. Integr8 is available at http://www.ebi.ac.uk/integr8.

Within-species sequence variation data are of special interest since they contain information about recent population/species history, and the molecular evolutionary forces currently in action in natural populations. These data, however, are presently dispersed within generalist databases, and are difficult to access. To solve this problem, we have developed Polymorphix, a database dedicated to sequence polymorphism. It contains within-species homologous sequence families built using EMBL/GenBank under suitable similarity and bibliographic criteria. Polymorphix is an ACNUC structured database allowing both simple and complex queries for population genomic studies. Alignments within families as well as phylogenetic trees can be download. When available, outgroups are included in the alignment. Polymorphix contains sequences from the nuclear, mitochondrial and chloroplastic genomes of every eukaryote species represented in EMBL. It can be accessed by a web interface (http://pbil.univ-lyon1.fr/polymorphix/query.php).

We analysed the length distributions of different types of beta-strand in a high resolution, non-homologous set of 500 protein structures, finding differences in their mean lengths. Antiparallel edge strands in strand-turn-strand motifs show a preference for an even number of residues. This propensity is enhanced if the length is corrected for beta-bulges, which insert an extra residue into the strand. Residues in antiparallel edge beta-strands alternate between being in hydrogen bonded and non-hydrogen bonded rings. Antiparallel edges with an even number of residues are more likely to have their final beta residue in a non-hydrogen bonded ring. This suggests that non-hydrogen bonded rings are intrinsically more stable than hydrogen bonded rings, perhaps because its side chain packing is closer. Therefore, we suggest that a simple way to increase beta-hairpin stability, or the stability of an antiparallel edge strand, is to have a non-hydrogen bonded ring at the end of the strand.

We have determined the crystal structure of YodA, an Escherichia coli protein of unknown function. YodA had been identified under conditions of cadmium stress, and we confirm that it binds metals such as cadmium and zinc. We have also found nickel bound in one of the crystal forms. YodA is composed of two domains: a main lipocalin/calycin-like domain and a helical domain. The principal metal-binding site lies on one side of the calycin domain, thus making YodA the first metal-binding lipocalin known. Our experiments suggest that YodA expression may be part of a more general stress response. From sequence analogy with the C-terminal domain of a metal-binding receptor of a member of bacterial ATP-binding cassette transporters, we propose a three-dimensional model for this receptor and suggest that YodA may have a receptor-type partner in E. coli.

The World Wide Web server of the PBIL (Pôle Bioinformatique Lyonnais) provides on-line access to sequence databanks and to many tools of nucleic acid and protein sequence analyses. This server allows to query nucleotide sequence banks in the EMBL and GenBank formats and protein sequence banks in the SWISS-PROT and PIR formats. The query engine on which our data bank access is based is the ACNUC system. It allows the possibility to build complex queries to access functional zones of biological interest and to retrieve large sequence sets. Of special interest are the unique features provided by this system to query the data banks of gene families developed at the PBIL. The server also provides access to a wide range of sequence analysis methods: similarity search programs, multiple alignments, protein structure prediction and multivariate statistics. An originality of this server is the integration of these two aspects: sequence retrieval and sequence analysis. Indeed, thanks to the introduction of re-usable lists, it is possible to perform treatments on large sets of data. The PBIL server can be reached at: http://pbil.univ-lyon1.fr.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues., (Copyright 2001 Wiley-Liss, Inc.)

N1 is the first residue in an alpha-helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH(3)CO-XAAAAQAAAAQAAGY-NH(2) at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp(-), Ala > Glu(-) > Glu(0) > Trp, Leu, Ser > Asp(0), Thr, Gln, Met, Ile > Val, Pro > Lys(+), Arg, His(0) > Cys, Gly > Phe > Asn, Tyr, His(+). N1 preferences are clearly distinct from preferences for the preceding N-cap and alpha-helix interior. pK(a) values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the alpha-helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 alpha-helix sites in proteins and in predicting the helix contents of peptides.

It is widely believed that the dominant force opposing protein folding is the entropic cost of restricting internal rotations. The energetic changes from restricting side-chain torsional motion are more complex than simply a loss of conformational entropy, however. A second force opposing protein folding arises when a side-chain in the folded state is not in its lowest-energy rotamer, giving rotameric strain. chi strain energy results from a dihedral angle being shifted from the most stable conformation of a rotamer when a protein folds. We calculated the energy of a side-chain as a function of its dihedral angles in a poly(Ala) helix. Using these energy profiles, we quantify conformational entropy, rotameric strain energy and chi strain energy for all 17 amino acid residues with side-chains in alpha-helices. We can calculate these terms for any amino acid in a helix interior in a protein, as a function of its side-chain dihedral angles, and have implemented this algorithm on a web page. The mean change in rotameric strain energy on folding is 0.42 kcal mol-1 per residue and the mean chi strain energy is 0.64 kcal mol-1 per residue. Loss of conformational entropy opposes folding by a mean of 1.1 kcal mol-1 per residue, and the mean total force opposing restricting a side-chain into a helix is 2.2 kcal mol-1. Conformational entropy estimates alone therefore greatly underestimate the forces opposing protein folding. The introduction of strain when a protein folds should not be neglected when attempting to quantify the balance of forces affecting protein stability. Consideration of rotameric strain energy may help the use of rotamer libraries in protein design and rationalise the effects of mutations where side-chain conformations change., (Copyright 2001 Academic Press.)

Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

We surveyed 299 high resolution, non-homologous protein crystal structures for alpha-helix lengths and capping preferences. We find that helices show a preference to have close to an integral number of turns. Helices can be usefully subdivided into either "favoured length" with 6, 7, 10, 11, 13, 14, 17, 18, 21, 22, 24, 25, 28, 29 or 31 residues, or "disfavoured length" with 8, 9, 12, 15, 16, 19, 20, 23, 26, 27 or 30 residues. Favoured length helices have their N and C-caps on the same side of the helix so they can lie on the protein surface. There is no significant difference in amino acid preferences at the N terminus between favoured and disfavoured length helices. At the C terminus, favoured length helices prefer non-polar side-chains at C4 and polar amino acid residues at C2, while disfavoured length helices prefer non-polar amino acid residues at C2. There are strong periodic trends in the likelihood of terminating a helix with a Schellman or alphaL C-capping motif. These can be rationalised by the preference for a non-polar side-chain at C3 with these motifs, favouring placing C3 on the buried side of the helix. We suggest that algorithms aiming to predict helices or C-capping in proteins should include a weight for the helix length., (Copyright 1999 Academic Press.)

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins., (Copyright 1999 Academic Press.)

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.