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At least 289 records · Page 16Linked to original sources
PubMed1994

Insertion in barnase of a loop sequence from ribonuclease T1. Investigating sequence and structure alignments by protein engineering.

Barnase was mutated by inserting into its active site loop sequences found in the related enzyme ribonuclease T1 (RNase T1), according to either structural or sequential similarity alignments. The barnase/RNase T1 hybrid corresponding to the structural alignment of the two proteins, endo-[RNaseT1-(93-99)]102abarnase, contains RNase T1 residues at positions 93-99 inserted between residues at positions 102 and 103 of barnase. The other constructed mutant, endo-[RNaseT1-(95-98)]104abarnase, has RNase T1 residues at positions 95-98 inserted between residues at positions 104 and 105 in barnase, corresponding to published sequence alignments of the two proteins in this region. The mutants were characterized by absorbance, fluorescence and CD spectroscopy; the stability, folding and unfolding kinetics, and catalytic activity were measured and compared with the wild-type enzyme. Endo-[RNaseT1-(93-99)]102abarnase, the mutant protein corresponding to the structural alignment of barnase with ribonuclease T1, shows a slightly higher stability (approximately 5 kJ/mol) towards urea and heat denaturation than the mutant endo-[RNaseT1-(95-98)]104abarnase, designed according to a sequence alignment between the two enzymes. Both mutants have very low catalytic activity, although the effect of mutation is almost entirely limited to kcat in the case of the mutant corresponding to the structural alignment between barnase and ribonuclease T1, while both kcat and Km are affected in the mutant corresponding to the sequence alignment between the two enzymes. Thus, the superiority of structural over sequential alignments cannot be supported conclusively by direct experiment in the present case.

Amino Acid Sequence↗
PubMed2001

Stabilization of short collagen-like triple helices by protein engineering.

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.

Amino Acid Sequence↗
PubMed1993

Rational protein engineering and industrial application: structure prediction by homology and rational design of protein-variants with improved 'washing performance'--the alkaline protease from Bacillus alcalophilus.

The successful attempt is presented to engineer an enzyme with respect to its technical application by the use of computer-aided protein design techniques. Based on a modeled 3-D structure a number of mutants of a subtilisin-like protease was designed with the aim to increase its washing performance. The model of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus was developed by the process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from the Brookhaven protein databank. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures. On the other hand, interesting deviations between the two structures were observed in some external loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of additional prolines and the selection of correct loops in deletion or insertion regions.

Amino Acid Sequence↗
PubMed2004

Engineered protein cages for nanomaterial synthesis.

Self-assembled particles of genetically engineered human L subunit ferritin expressing a silver-binding peptide were used as nanocontainers for the synthesis of silver nanoparticles. The inner cavity of the self-assembled protein cage displays a dodecapeptide that is capable of reducing silver ions to metallic silver. This chimeric protein cage when incubated in the presence of silver nitrate exhibits the growth of a silver nanocrystal within its cavity. Our studies indicate that it is possible to design chimeric cages, using specific peptide templates, for the growth of other inorganic nanoparticles.

Amino Acid Sequence↗
PubMed1993

Rationally designing the accumulation of a folding intermediate of barnase by protein engineering.

A method for the stabilization of transient folding intermediates is presented. Barnase folds and unfolds via such an intermediate. Mutations that destabilize the folded state relative to the folding intermediate had been previously identified from the free energy profiles for the unfolding of mutant proteins. It is predicted that the accumulation of such mutations should lead to the intermediate being the most stable species at certain concentrations of denaturant. Mutants were prepared that contained combinations of such mutations. The behavior of these mutants on urea denaturation was studied by probes for tertiary structure (fluorescence, near-UV CD), secondary structure (far-UV CD), and hydrodynamic volume (size-exclusion chromatography). Whereas wild-type shows a two-state transition in all cases, with the same thermodynamic values being found by all probes, some of the mutants show different transitions with different structural probes. On increasing concentration of denaturant, the tertiary structure of these mutants is lost before all the secondary structure and before the protein shows the maximum expanded volume that is characteristic of the unfolded state. These mutants thus accumulate an intermediate state at equilibrium under certain urea concentrations. The intermediate state retains some degree of secondary structure but has a disrupted tertiary structure, and its degree of compactness is intermediate between the folded and the unfolded forms, probably expanding with increasing concentration of denaturant. The accumulation of the intermediate should allow its direct characterization by spectroscopy, especially NMR.

Bacterial Proteins↗
PubMed2005

Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics.

Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems.

Calcium↗
PubMed1989

Conformation of beta-hairpins in protein structures. A systematic classification with applications to modelling by homology, electron density fitting and protein engineering.

A systematic classification of beta-hairpin structures which takes into account the polypeptide chain length and hydrogen bonding between the two antiparallel beta-strands is described. We have used this classification of beta-hairpin structures and their specific sequence pattern to derive rules which demonstrate its usefulness in assisting modelling beta-hairpins. These rules can be applied to comparative model building, modelling into electron density and in the prediction of conformation of beta-hairpins to aid protein engineering.

Aspartic Acid Endopeptidases↗
PubMed1995

Protein fragments as models for events in protein folding pathways: protein engineering analysis of the association of two complementary fragments of the barley chymotrypsin inhibitor 2 (CI-2).

Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of association of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies of the binding of fragments and from thermal denaturation of the complexes. In general, there is a good correlation between the changes on mutation of the free energy of association of fragments and the changes in free energy of folding of the uncleaved parent protein. The notable exceptions are for residues in regions of the fragments that form nonnative hydrophobic clusters in the isolated fragments; mutation of the hydrophobic residues involved in these clusters decreases the equilibrium constant for formation of the noncovalent complex less than it does the equilibrium constant for folding of intact protein. The dissociated fragments must be destabilized by mutation of those hydrophobic residues, but to a lesser extent than is the complex itself. These clusters are thus less important energetically in the denatured state of the intact protein. The second-order rate constants for the major phase of association change with mutation, similar results being obtained from fluorescence measurements of the regain of tertiary structure and from circular dichroism measurements of the regain of secondary structure. The rate constants for association correlate well, in general, with the rate constants of refolding of the respective uncleaved proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1996

Protein engineering studies of dichloromethane dehalogenase/glutathione S-transferase from Methylophilus sp. strain DM11. Ser12 but not Tyr6 is required for enzyme activity.

The structural gene for dichloromethane dehalogenase/glutathione S-transferase (GST, EC 2.5.1.18) from Methylophilus sp. strain DM11 was subcloned into a multicopy plasmid under the control of the T7 polymerase promoter, allowing expression in Escherichia coli and easy purification of the enzyme in good yield. Several point mutations leading to amino acid changes at residues Tyr6, His8 and Ser12 of the protein were introduced in this gene. Mutations at Tyr6, the N-terminal tyrosine known to be essential for enzymatic activity in glutathione S-transferases of the alpha, mu, and pi classes, had little effect on the activity of dichloromethane dehalogenase. The same applied for mutations at residue His8, which from multiple alignments of GST sequences may also correspond to the conserved N-terminal tyrosine residue of GST enzymes. The higher turnover rate of the wild-type enzyme with dibromomethane compared with dichloromethane was lost in mutants with amino acid replacements at residue His8, but retained in mutant proteins at Tyr6. Mutations at Ser12 led to mutants with drastically reduced enzymatic activity, pinpointing this residue as an essential determinant of catalytic efficiency.

Amino Acid Sequence↗
PubMed1988

Channel protein engineering: synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers.

A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nonselective between Na+ and K+. However, the synthetic channel displays no significant voltage dependence. Energetic considerations suggest a bundle of four parallel amphipathic alpha-helices as the most plausible channel structure. The synthetic 22-mer channel-forming peptide allows study of the mechanisms of ion permeation through sodium channels by protein engineering techniques.

Amino Acid Sequence↗
PubMed1994

Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability.

Most starch hydrolases and related enzymes belong to the alpha-amylase family which contains a characteristic catalytic (beta/alpha)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the alpha-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the alpha-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the beta-->alpha loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of alpha-amylases, changed the distribution of alpha-, beta- and gamma-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative alpha-1,4:alpha-1,6 dual-bond specificity of neopullulanase. Barley alpha-amylase isozyme hybrids and Bacillus alpha-amylases demonstrate the impact of a small domain B protruding from the (beta/alpha)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as alpha-amylase, starch branching and debranching enzymes, and amylomaltase.

Amino Acid Sequence↗
PubMed1985

Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering.

The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the alpha-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the gamma-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine-ATP] transition state [the mutant tyrosyl-tRNA synthetase (Thr-40----Ala-40; His-45----Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 X 10(5) but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accomplished by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.

Adenosine Triphosphate↗
PubMed1986

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers.

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.

Amino Acyl-tRNA Synthetases↗