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At least 325 records · Page 18Linked to original sources
PubMed2002

Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal gamma-haemolysins in lipid membranes.

Staphylococcal gamma-haemolysins are bicomponent toxins in a family including other leucocidins and alpha-toxin. Two active toxins are formed combining HlgA or HlgC with HlgB. Both open pores in lipid membranes with conductance, current voltage characteristics and stability similar to alpha-toxin, but different selectivity (cation instead of anion). Structural analogies between gamma-haemolysins and alpha-toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in alpha-toxin, but either positive or negative in gamma-haemolysins. Four mutants were produced (HlgA D44K, HlgB D47K, HlgB D49K and HlgB D47K/D49K) converting those negative charges to positive in HlgA and HlgB. When all charges were positive, the pores had the same selectivity and conductance as alpha-toxin, suggesting that the cluster may form an entrance electrostatic filter. As mutated HlgC-HlgB pores were less affected, additional charges in the lumen of the pore were changed (HlgB E107Q, HlgB D121N, HlgB T136D and HlgA K108T). Removing a negative charge from the lumen made the selectivity of both HlgA-HlgB D121N and HlgC-HlgB D121N more anionic. Residue D121 of HlgB is compensated by a positive residue (HlgA K108) in the HlgA-HlgB pore, but isolated in the more cation-selective HlgC-HlgB pore. Interestingly, the pore formed by HlgA K108T-HlgB, in which the positive charge of HlgA was removed, was as cation selective as HlgC-HlgB. Meanwhile, the pore formed by HlgA K108T-HlgB D121N, in which the two charge changes compensated, retrieved the properties of wild-type HlgA-HlgB. We conclude that the conductance and selectivity of the gamma-haemolysin pores depend substantially on the presence and location of charged residues in the channel.

Amino Acid Sequence↗
PubMed2005

Simulation modeling of pooling for combinatorial protein engineering.

Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, betagalactosidase (supermutant, or greatly improved enzyme) and beta-glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Additional simulations using more complex activity distributions show the versatility of the model. Pooling is most suited to cases such as the directed evolution of new function in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling is most successful when a sensitive assay is employed. Using the model will increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.

Cells, Cultured↗
PubMed2005

Protein engineering of the archetypal nitroarene dioxygenase of Ralstonia sp. strain U2 for activity on aminonitrotoluenes and dinitrotoluenes through alpha-subunit residues leucine 225, phenylalanine 350, and glycine 407.

Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the alpha-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.

Dioxygenases↗
PubMed1997

Identification of elements critical for phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by adenosine monophosphate-activated protein kinase: protein engineering of the naturally nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pseudomonas mevalonii.

The initially nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii (E.C. 1.1.1.88) was engineered to phosphorylatable forms in order to identify elements critical for phosphorylation of HMG-CoA reductase by AMP-activated protein kinase. P. mevalonii, mutant enzymes phosphorylatable by AMP-activated protein kinase were engineered by substituting cognate residues from the kinase recognition sequence of Syrian hamster HMG-CoA reductase (E.C. 1.1.1.34). Various combinations of residues 381-391, which correspond to the kinase recognition sequence of the hamster enzyme, were mutated. P. mevalonii mutant enzyme R387S, in which a serine had been inserted at position P, which corresponds to that of the regulatory serine of the hamster enzyme, was only weakly phosphorylated. Genes that encoded thirty-six additional mutant enzymes containing various portions of the hamster kinase recognition sequence were constructed. Following expression, purified mutant enzymes were assayed as substrates for AMP-activated protein kinase. Identified as critical for phosphorylation was the simultaneous presence of aspartate or asparagine at position P+3 and of leucine at position P+4, three and four residues on the C-terminal side of the phosphorylatable serine, respectively. Two basic residues at positions P-1, P-2, or P-3 also appeared to be critical for phosphorylation when present in combination with aspartate or asparagine at P+3 and leucine at P+4.

AMP-Activated Protein Kinases↗
PubMed1994

Reconstruction of ancestral sequences by the inferential method, a tool for protein engineering studies.

This paper describes the inferential method, an approach for reconstructing protein and nucleotide sequences of ancestral species, starting from known, homologous, contemporary sequences. The method requires knowledge of the topology of the phylogenetic tree, whose nodes are the species to whom the reconstructed sequences belong. The method has been tested by computer simulation of speciation and nucleotide substitutions, starting from a single ancestral sequence, and by subsequent reconstruction of nodal sequences. Results have shown that reconstructions obtained by the inferential method are affected by limited error frequencies, which (1) are proportional to the squares of nucleotide substitution rates and of internodal distances, and (2) are little influenced by non-uniformity of transformation rates of nucleotides. Furthermore, good agreement of the results has been obtained by comparing protein-sequence reconstructions carried out with the inferential method with those obtained using the maximum parsimony method in two different cases: e.g., a reconstruction of simulated sequences and a reconstruction of mammalian ribonuclease sequences.

Amino Acid Sequence↗
PubMed2001

Optical determination of glutamine using a genetically engineered protein.

We have developed a reagentless optical biosensor for glutamine based on the Escherichia coli glutamine binding protein (GlnBP). Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes. The fluorescence emission of acrylodan and 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35%, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 microM; K(Dapp) 160 nM). No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine, and arginine. Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GlnBPS179C-acrylodan showed a 13-ns rotational correlation time in the ligand-free state, whereas multiple correlation times were assigned in the glutamine-bound conformation. The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine. The engineered GlnBP is a first step toward the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures.

Biosensing Techniques↗
PubMed1991

Mapping transition states of protein unfolding by protein engineering of ligand-binding sites.

We describe a method for probing the integrity and relative orientation of structural elements that are indirectly linked by ligands in protein complexes during protein folding. The effect of 3'-GMP on the rate constants of unfolding of wild-type barnase and several mutants has been studied. By comparing the rates of unfolding of wild-type and mutant proteins, we show that the interaction between His102 and 3'-GMP is fully retained in the transition state compared with the folded state, while the interaction between Glu60 and the ligand is partly retained and that of Lys27 is broken. Our data suggest that the transition state has a partly formed ligand binding site in which the guanine binding loop containing Glu60 and the loop containing His102 are formed at the sides of the beta-sheet but the docking of the N terminus of the second alpha-helix containing Lys27 on the beta-sheet is disrupted. The active site of barnase in complexes is thus partly retained in the transition state of unfolding. Although the ligand could in principle perturb the unfolding pathway, there is independent evidence that indicates that similar structural changes occur upon unfolding of unligated barnase.

Bacterial Proteins↗
PubMed1990

Protein engineering for thermostability.

Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins.

Amino Acid Sequence↗
PubMed1991

Artificial cell adhesive proteins engineered by grafting the Arg-Gly-Asp cell recognition signal: factors modulating the cell adhesive activity of the grafted signal.

An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.

Amino Acid Sequence↗
PubMed1992

The organisation of production of genetically-engineered proteins in yeast.

The use of a variety of genetic-engineering techniques to introduce foreign DNA into living yeast cells has resulted in the production in these cells of the corresponding proteins. These include enzymes, antibodies, vaccines, anti-viral agents, and hormones. This article discusses the techniques of genetic engineering in yeast and suggests the lines of future progress in the economic production of novel proteins for use in therapy.

Plasmids↗
PubMed1995

Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.

Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady-state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.

Amino Acid Sequence↗
PubMed2005

Exploring amino-acid radical chemistry: protein engineering and de novo design.

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.

Amino Acids↗