Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

A new look at an old virus: patterns of mutation accumulation in the human H1N1 influenza virus since 1918

BACKGROUND: The H1N1 influenza A virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917–1918. Initial mortality was extremely high, but dropped exponentially over time. Influenza viruses have high mutation rates, and H1N1 has undergone significant genetic changes since 1918. The exact nature of H1N1 mutation accumulation over time has not been fully explored. METHODS: We have made a comprehensive historical analysis of mutational changes within H1N1 by examining over 4100 fully-sequenced H1N1 genomes. This has allowed us to examine the genetic changes arising within H1N1 from 1918 to the present. RESULTS: We document multiple extinction events, including the previously known extinction of the human H1N1 lineage in the 1950s, and an apparent second extinction of the human H1N1 lineage in 2009. These extinctions appear to be due to a continuous accumulation of mutations. At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). The accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. We observed a continuous erosion over time of codon-specificity in H1N1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns. CONCLUSIONS: While there have been numerous adaptations within the H1N1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. We suggest H1N1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. These findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide

A transgenic plant having enhanced drought tolerance

The present invention relates to the field of transgenic plants with novel phenotypes, especially plants with enhanced drought and pathogen resistance. Provided are transgenic crop plants comprising integrated in their genome a chimeric gene, characterized by said chimeric gene comprising a transcription regulatory sequence active in plant cells operably linked to a nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 3 or a protein at least 70 % identical to SEQ ID NO: 3, or an ortholog protein or a functional fragment thereof. In addition to enhanced drought tolerance the transgenic plants may show enhanced disease resistance and enhanced root structure

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.Proteiinien muokkauksella tähdätään entsyymien ja sitojareagenssien ominaisuuksien parantamiseen geneettisten muutosten avulla. Tyypillisiä parannettavia ominaisuuksia ovat sitomisvoimakkuus, spesifisyys, kestävyys, tuotto-ominaisuudet ja liukoisuus. Koska proteiinit ovat monimutkaisia biomolekyylejä, geneettisten muutosten vaikutukset ovat vain harvoin tarkkaan ennustettavissa. Siksi suosittu proteiinien muokkausstrategia on luoda kohdemolekyylistä lukuisia geenivariantteja ja asettaa luotu joukko valintakokeeseen, jonka perusteella variantit erottuvat toisistaan tavoitellun ominaisuuden perusteella. Tämä suunnattuna evoluutiona tunnettu tekniikka on keskeinen työkalu kehitettäessä proteiinituotteita, joita käytetään monenlaisissa sovelluksissa vaatteiden pesuaineista syöpälääkkeisiin. Proteiinituotteiden kehitystyön nopeuttaminen edellyttää jatkuvasti uusia menetelmiä, joilla voidaan rakentaa aiempaa laajempia geenikirjastoja ja niille yhteensopivia valintatyökaluja. Tässä työssä tutkittiin alukepidennysmutageneesitekniikan mahdollisuuksia korkealaatuisempien geenivarianttikirjastojen rakentamiseksi Escherichia coli-bakteerin soluihin. Toisessa osajulkaisussa tutkittiin rekombinaatiota menetelmänä, jolla voitaisiin lisätä seulottavissa olevien entsyymivarianttien lukumäärää. Kolmannessa osajulkaisussa kehitettiin valintaprosessi, jonka tarkoituksena oli parantaa vastaainefragmenttien ilmentymistä filamenttifaagin pinnalla näyttötekniikkaa varten. Neljännessä osajulkaisussa testattiin geenikirjaston suunnittelustrategioita käytännössä kahdella eri periaatteiden mukaan monimuotoistetulla vasta-ainekirjastolla. Viimeisessä osajulkaisussa vertailtiin faagin eri pintaproteiineihin fuusioidun vasta-ainekirjaston toimintaa valintakokeiden avulla. Samalla tutkittiin vasta-ainefragmenttien Fab (engl. antigen binding fragment) ja ScFv (engl. single-chain fragment of antibody variable domains) vaikutusta valintakokeen onnistumiseen, jotta selviäisi, mikä on käyttökelpoisin näyttötekniikka käytössä olevan kirjaston hyödyntämiseksi. Tutkimuksen tuloksena kehitettiin uusi geenivarianttikirjaston rakennusmenetelmä nimeltään sRCA (engl. selective rolling circle amplification), joka lisää merkittävästi mutageneesitehokkuutta ja jopa yli satakertaistaa kirjaston muodostavien transformanttien määrän verrattuna tavanomaiseen alukepidennysmutageneesiin. Toisessa osajulkaisussa havaittiin Cre-loxP-rekombinaation olevan sovelias työkalu RCA-satunnaismutageneesin tuloksena syntyvän DNA-vyyhdin pilkkomiseen kehämäisiksi plasmidiyksiköiksi. Uusi menetelmä lisäsikin DNA:n transformaatiotehokkuutta E. coli-bakteeriin. Kirjastoseulontojen avulla osoitettiin, että kirjastosta, jossa aminohappojen vaihtelua esiintyi lähempänä vasta-aineen sitomiskohdan keskustaa, löytyi enemmän pienmolekyylejä ja peptidejä tunnistavia vasta-aineita. Ulompana keskustasta sijaitsevien aminohappojen vaihtelu puolestaan tuki proteiineja tunnistavien vasta-aineiden kehitystyötä. Vasta-ainekirjaston faaginäyttötekniikkavertailun johtopäätökset olivat, että käyttämällä filamenttifaagin vasta-aineiden fuusiokumppanina lyhennettyä proteiini kolmea (p3Δ) pystyttiin eristämään runsaammin erilaisia kohdetta tunnistavia vasta-ainemolekyylejä kuin käyttämällä kokopitkää proteiini kolmea tai proteiini yhdeksää (p9). Erityisesti ScFvp3Δ- formaatissa oleva kirjasto tarjosi sitomisvoimakkuudeltaan parhaita vasta-aineita molekyylikooltaan pienen digoksigeniinin tunnistukseen. Tutkimus geenivarianttikirjastojen suunnittelusta, rakentamisesta ja seulontaan soveltuvista valintatyökaluista lisää käytännön tietoa suunnattujen evoluutiokokeiden toteuttamiseksi ja on arvokasta tietoa alan tutkijoille heidän tulevissa hankkeissaan.Siirretty Doriast