Channel protein engineering. An approach to the identification of molecular determinants of function in voltage-gated and ligand-regulated channel proteins.
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Amino Acid Sequence↗
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Results for “Protein Engineering”
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Nucleic acid synthesis and protein engineering.
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Genetic Engineering↗
Immunologic and Ultrastructural Characterization of HIV Pseudovirions Containing Gag and Env Precursor Proteins Engineered in Insect Cells
Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.
Journal Article↗
Potential of genetic algorithms in protein folding and protein engineering simulations.
Genetic algorithms are very efficient search mechanisms which mutate, recombine and select amongst tentative solutions to a problem until a near optimal one is achieved. We introduce them as a new tool to study proteins. The identification and motivation for different fitness functions is discussed. The evolution of the zinc finger sequence motif from a random start is modelled. User specified changes of the lambda repressor structure were simulated and critical sites and exchanges for mutagenesis identified. Vast conformational spaces are efficiently searched as illustrated by the ab initio folding of a model protein of a four beta strand bundle. The genetic algorithm simulation which mimicked important folding constraints as overall hydrophobic packaging and a propensity of the betaphilic residues for trans positions achieved a unique fold. Cooperativity in the beta strand regions and a length of 3-5 for the interconnecting loops was critical. Specific interaction sites were considerably less effective in driving the fold.
Algorithms↗
Why ion pair reversal by protein engineering is unlikely to succeed.
Genetic engineering is a powerful tool for exploring correlations between structure and function in proteins, but as yet we are unable to use it for effective protein design. One of the most interesting examples, which would seem to be obvious, is reversing the polarity of an ion pair. Changing a positively charged protein group, that provides a strong binding for negative substrates, to a negative group is expected to provide an effective binding site for a positively charged substrate. But several recent experiments on aspartate aminotransferase, trypsin and aspartate transcarbamoylase (Schachman, H. K. personal communication) have indicated that polarity reversal is not so successful. Here we argue that the same factors that make the enzyme an effective system for the (-+) pair will make it a much less effective system for the (+-) pair. We also point out that the unusually low effective dielectric constant (epsilon approximately equal to 13) for the (-+) interaction is due to its microenvironment and this will destabilize a (+-) arrangement having an entirely different dielectric constant (epsilon approximately equal to 80). The calculations presented here evaluate the energetics of ion pairs in protein active sites on a semiquantitative level. This is particularly important when dealing with strong, functionally important interactions that are difficult to evaluate with macroscopic models.
Aspartate Aminotransferases↗
[Prognosis of the degree of exposure of amino acid residues in globular proteins in relation to various problems of protein engineering].
The algorithm was developed to predict the degree of exposure of amino acid residues in globular proteins. This algorithm combined with standard discriminant analysis methods was used for evaluation of the accessibility of Lys and Arg residues for trypsin-like proteases attack. The procedure can be useful for a computer-aided design of prolonged-action protein drug preparations.
Amino Acids↗
Bioinformatics-driven, rational engineering of protein thermostability.
A longstanding goal in protein engineering is to identify specific sequence changes that endow proteins with desired functional properties. As opposed to traditional rational and random protein engineering techniques, we have employed a bioinformatic approach to identify specific sequence changes that influence key functional properties of a protein within a defined superfamily. Specifically, we have used the Bayesian sequence-based algorithms PROBE and Classifier to identify a strand-turn-strand motif that contributes to thermophilicity among members of the serine protease subtilase superfamily. By replacing a 16 amino acid sequence in the mesophilic subtilisin E (from Bacillus subtilis) with a bioinformatics-generated thermophilic model sequence, the melting temperature of subtilisin E was increased by 13 degrees C. While wild-type subtilisin E was inactive at 90 degrees C, the mutant retained a substantial fraction of its function, with ca. one-third of the activity that it has at 45 degrees C.
Algorithms↗
Engineered protein scaffolds for molecular recognition.
The use of so-called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine. This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of peptide segments that are hypervariable both in sequence and in conformation. After the application of antibody engineering methods along with library techniques had resulted in first successes in the selection of functional antibody fragments, several laboratories began to exploit other types of protein architectures for the construction of practically useful binding proteins. Properties like small size of the receptor protein, stability and ease of production were the focus of this work. Hence, among others, single domains of antibodies or of the immunoglobulin superfamily, protease inhibitors, helix-bundle proteins, disulphide-knotted peptides and lipocalins were investigated. Recently, the scaffold concept has even been adopted for the construction of enzymes. However, it appears that not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand-binding sites with high affinities and specificities. This review will therefore concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity. An overview will be provided about the current approaches, and some emerging trends will be identified. (c) 2000 John Wiley & Sons, Ltd. Abbreviations used: ABD albumin-binding domain of protein G APPI Alzheimer's amyloid beta-protein precursor inhibitor BBP bilin-binding protein BPTI bovine (or basic) pancreatic trypsin inhibitor BSA bovine serum albumin CBD cellulose-binding domain of cellobiohydrolase I CD circular dichroism Cdk2 human cyclin-dependent kinase 2 CDR complementarity-determining region CTLA-4 human cytotoxic T-lymphocyte associated protein-4 FN3 fibronectin type III domain GSH glutathione GST glutathione S-transferase hIL-6 human interleukin-6 HSA human serum albumin IC(50) half-maximal inhibitory concentration Ig immunoglobulin IMAC immobilized metal affinity chromatography K(D) equilibrium constant of dissociation K(i) equilibrium dissociation constant of enzyme inhibitor LACI-D1 human lipoprotein-associated coagulation inhibitor pIII gene III minor coat protein from filamentous bacteriophage f1 PCR polymerase-chain reaction PDB Protein Data Bank PSTI human pancreatic secretory trypsin inhibitor RBP retinol-binding protein SPR surface plasmon resonance TrxA E. coli thioredoxin
Animals↗
Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering.
The Bacillus licheniformis and Bacillus amyloliquefaciens subtilisins differ by 31% in protein sequence and by factors of greater than 60 in catalytic efficiency, kcat/Km, toward various substrates. Despite large differences in sequence and substrate specificity for these serine proteases, only two amino acid substitutions (residues 156 and 217) occur within 4 A (contact distance) of modeled substrates, and a third substitution (residue 169) is within 7 A. The three B. licheniformis substitutions (Ser-156/Ala-169/Leu-217) were introduced into the wild-type B. amyloliquefaciens subtilisin (Glu-156/Gly-169/Tyr-217) by site-directed mutagenesis. The substrate specificity of the triple mutant approaches that of B. licheniformis enzyme when assayed with seven different substrates that vary in charge, size, and hydrophobicity. Thus, specificity properties of distantly related and functionally divergent enzymes can be exchanged by limited amino acid replacements, in this case representing less than 4% of the sequence differences.
Amino Acids↗
Computer aided peptide and protein engineering.
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Algorithms↗
Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates.
According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.
Asparagine↗
Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii. Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation.
The activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif. We have engineered P. mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation. We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes. A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L). The activity of P. mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation. Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase. Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity. Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity. As in the analogous Syrian hamster mutant enzyme S871D, P. mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge. Engineering of P. mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies. Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P. mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.
Animals↗
A protein sequence/structure database. Protein Engineering Club Database Group.
Researchers have been wishing for years for a database integrating protein sequences and protein structures. A group from the United Kingdom has achieved this long-sought objective.
Amino Acid Sequence↗
Evolution of haemoglobin studied by protein engineering.
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Animals↗
Gene transfer into CD4+ T lymphocytes: green fluorescent protein-engineered, encephalitogenic T cells illuminate brain autoimmune responses.
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Animals↗
Conformation-directed recombination of enzyme-activated peptide fragments: a simple and efficient means to protein engineering. Its use in the creation of cytochrome c analogues for structure-function studies.
Protein fragments have been activated by the addition of amino acid esters using proteolytic enzymes under conditions where the equilibria are shifted in the direction of synthesis. Because of the natural propensity of large protein fragments to form complexes approximating native conformation, these activated fragments have been induced to recombine by formation of the missing peptide bond. Although the incorporated ester is only weakly activating, the complex, mimicking an enzyme, provides proximity and orientation at the reacting termini, so that coupling yields are high. In other words, the protein catalyzes its own resynthesis. What distinguishes our technique from the rare natural examples of this phenomenon is that it operates at a variety of cleavage sites and with varying chain lengths. There seems to be no particular limitations on the amino acid esters that can be added, and serine proteases with a wide range of specificities can be used. It thus appears that we have a truly general method for the condensation of large fragments in protein synthesis, be they natural or the products of synthetic or genetic methods. This approach has the advantages over conventional methods of great specificity, high efficiency, and mild conditions of use. With our model protein, cytochrome c, we have used this approach to make analogues that illuminate structure-function relations. Both the highly conserved lysine 39 and the functionally invariant threonine 40 have been replaced by a range of substitutions. The results show how crucial these residues are to the structural and functional integrity of the bottom omega-loop of the protein.
Cyanogen Bromide↗
Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices.
Amino acid sequences have been compared for thermophilic and mesophilic molecules from six different protein families, which include lactate and glyceraldehyde-3-phosphate dehydrogenases, triose phosphate isomerases, superoxide dismutases, thermolysins and subtilisins. Since a three-dimensional structure was known for at least one of the sequences in each family, analysis of preferred residue substitutions, presumably to achieve thermal stability, could be examined from a structural context. The overall results, which are generally consistent across all the families, suggested decreased flexibility and increased hydrophobicity in alpha-helical regions as the main stabilizing principles. The most favoured residual exchanges, hopefully useful in engineering stability into proteins, are discussed.
Amino Acid Sequence↗
Regulation of ribozyme activity by engineered protein switch.
Previously, we showed that the P3 domain of the Escherichia coli ribonuclease P ribozyme can be truncated and replaced in vitro. In this study, we prepared a P3-replaced variant of the E. coli ribozyme that has HIV TAR sequence as the engineered P3 domain. The mutant ribozyme demonstrated the ribonuclease P activity and was inhibited in the presence of the HIV tat protein fragment. Our results showed that the P3 domain of this enzyme can be engineered, and addition of some heterologous protein subunits can also be done to this domain.
Escherichia coli Proteins↗