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PubMed1987

Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships.

The activity of mutant enzymes can be analyzed quantitatively by structure-activity relationships in a manner analogous to Brønsted or Hammett plots for simple organic reactions. The slopes of such plots, the beta values, indicate for the enzymatic reactions the fraction of the overall binding energy used in stabilizing particular complexes. In particular, information can be derived about the interactions between the enzyme and the transition state. The activities of many mutant tyrosyl-tRNA synthetases fit well simple linear free energy relationships. The formation of enzyme-bound tyrosyl adenylate (E.Tyr-AMP) from enzyme-bound tyrosine and ATP (E.Tyr-ATP) results in an increase in binding energy between the enzyme and the side chain of tyrosine and the ribose ring of ATP. Linear free energy plots of enzymes mutated in these positions give the fraction of the binding energy change that occurs on formation of the transition state for the chemical reaction and the various complexes. It is shown that groups that specifically stabilize the transition state of the reaction are characterized by beta values much greater than 1. This is found for residues that bind the gamma-phosphate of ATP (Thr-40 and His-45) and have previously been postulated to be involved in transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1998

Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity.

Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis [Nath, K. (1981) Arch. Biochem. Biophys, 212, 611-617]. Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface. The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis. In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis. The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing. The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined. The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M). These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions. The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure.

Alanine↗
PubMed1992

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

Actinomycetales↗
PubMed1985

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering.

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.

Cloning, Molecular↗
PubMed1987

Natural selection, protein engineering, and the last riboorganism: rational model building in biochemistry.

A detailed study of the chemical behavior of modern catalysts (here, exemplified by dehydrogenases dependent on NAD+) allows us to construct models that distinguish between selected and drifting behaviors in biological macromolecules. These models enable us to manipulate rationally the properties of enzymes, here to design an "acetaldehyde reductase" dependent on NAD+ that is faster than any given us by nature. When applied to the origin of protein catalysis, models that explain the structures of ribo-cofactors (e.g., NAD+) must postulate a metabolically complex breakthrough organism. This means that: (1) The view from the present day back to the truly primeval organism is obscured; it is futile to try to deduce the detailed structure of the first life by examining the behaviors of modern organisms. (2) Riboorganisms dominated life on earth for a long time before translation evolved; indeed, fossils of riboorganisms might already be known. (3) Using organic synthesis, we have expanded the number of bases available for making RNA and making accessible RNA molecules that are likely to be intrinsically better catalysts.

Animals↗
PubMed1989

Protein engineering of disulfide bonds in subtilisin BPN'.

Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also possible that the rate of irreversible inactivation depends upon the kinetics and not the thermodynamics of unfolding and so the entropically stabilizing effect expected from a disulfide bond may not apply.

Bacillus↗
PubMed1996

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity.

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.

Animals↗
PubMedDate not supplied

Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering.

A detailed description of the interactions between an antibody and its epitope is necessary to allow an understanding of the way in which antibodies bind to antigenic surfaces presented by foreign molecules. Ideally this should be done by analysis of crystal structures of antibody-antigen complexes, but so far only two of these are available. An alternative strategy combines molecular modelling with site-directed mutagenesis (SDM) and using this we have generated a preliminary model of the complex between Gloop2, an antibody raised against a peptide containing the 'loop' determinant of hen egg-white lysozyme (HEL) which also binds the native protein, and its epitope on the protein surface. The main predictions from our model were; (1) that the surface of interaction between the antibody and the antigen is large (20 A X 15 A) and involves all the complementarity-determining regions (CDRs), (2) that electrostatic interactions were important in the formation of the complex, and (3) that conformational changes in either the loop or in the CDRs may occur during the formation of the complex. Here we report SDM studies which test some of these predictions; removal of two charged residues at the periphery of the combining site increases the affinity of the antibody for its antigen over 8-fold and decreases its ability to cross-react with closely-related antigens. This result is at variance with our original prediction but can be accommodated within our newly refined model; the role of electrostatics in antigen-antibody interactions is now questionable.

Animals↗
PubMed1987

Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering.

The dielectric constant in the active site cleft of subtilisin from Bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pKa value of the active site histidine (His64) by kinetics. Mutation of a negatively charged surface residue, which is 12 to 13 A from His64, to an uncharged one Asp----Ser99) lowers the pKa of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 M). This corresponds to an apparent dielectric constant of about 40 to 50 between Asp99 and His64. The mutation is in an external loop that is known to tolerate a serine at position 99 from homologies with subtilisins from other bacilli. The environment between His64 and Asp99 is predominantly protein. Another charged residue that is at a similar distance from His64 (14 to 15 A) and is also in an external loop that is known to tolerate a serine residue is Glu156, at the opposite side of the active site. There is only water in a direct line between His64 and Glu156. Mutation of Glu----Ser156 also lowers the pKa of His64 by up to 0.4 unit at low ionic strength. This change again corresponds to an apparent dielectric constant of about 40 to 50. The pKa values were determined from the pH dependence of kcat/KM for the hydrolysis of peptide substrates, with a precision of typically +/- 0.02 unit. The following suggests that the changes in pKa are real and not artefacts of experimental conditions: Hill plots of the data for pKa determination have gradients (h) of -1.00(+/- 0.02), showing that there are negligible systematic deviations from theoretical ionization curves involving a monobasic acid: the pH dependence for the hydrolysis of two different substrates (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide and benzoyl-L-valyl-L-glycyl-L-arginyl p-nitroanilide) gives identical results so that the pKa is independent of substrate; the pH dependence is unaffected by changing the concentration of enzyme, so that aggregation is not affecting the results; the shift in pKa is masked by high ionic strength, as expected qualitatively for ionic shielding of electrostatic interactions.

Amino Acid Sequence↗
PubMed1990

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, X. Analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from B. stearothermophilus and B. caldolyticus by protein engineering.

The amino-acid sequences of the lactate dehydrogenases (LDH) from B. stearothermophilus and B. caldolyticus differ at only 10 positions. The properties of these enzymes however show substantial differences. The LDH from B. stearothermophilus is activated by Fru-P2 and has a higher thermostability (10 degrees C) than the enzyme from B. caldolyticus which cannot be activated by Fru-P2. To correlate these functional differences to the structural properties, we have constructed a set of hybrid- and point-mutants of the two LDHs. The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes. This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins.

Amino Acid Sequence↗
PubMed1998

Protein engineering of ribonucleases.

Natural bovine seminal RNase possesses a potent antitumor action. We have mutagenized monomeric bovine pancreatic RNase A, devoid of any cytotoxic action, to insert residues present at corresponding positions in the subunit of dimeric, antitumor, seminal RNase. Like naturally dimeric seminal RNase, the mutant dimeric RNases display selective toxicity for malignant cells, which is absent in the monomeric mutants.

Amino Acid Substitution↗
PubMed2003

Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations.

Twelve mutations were constructed to improve the thermostability of glucoamylase from Aspergillus awamori based on the results of molecular dynamics simulations. The thermal unfolding of the catalytic domain followed a putative hierarchical behavior. In addition, the unfolding of the 13 alpha-helices obeyed the random ordered mechanism, in which the alpha-helices 8, 1 and 11 unfolded more rapidly than the others. The catalytic center was well protected by the (alpha/alpha)(6)-barrel at simulation temperatures up to 600 K, whereas the catalytic base, E400, migrated from its original interior pocket to the surface of the catalytic domain by surmounting the hydrophobic barrier provided by alpha-helices 12 and 13 at 800 K. The disulfide bonds engineered to 'lock' the alpha-helix 11 on the surface of the catalytic domain dramatically increased the thermostability. Substituting G396 and G407 with Ala residues slightly increased the thermostability, whereas their specific activity and catalytic efficiency were reduced. This indicates that the introduced residues with higher hydrophobicity were favorable in the loop between alpha-helices 12 and 13, whereas they partially destroyed the hydrogen bond and salt linkage network in the catalytic center. Alpha-helices 12 and 13 can be stabilized by introducing residues with higher hydrophobicity, except for the H391M mutation.

Amino Acid Substitution↗
PubMed1998

The enhancing of a cysteine proteinase activity at acidic pH by protein engineering, the role of glutamic 50 in the enzyme mechanism of caricain.

Carica papaya produces four cysteine proteinases. Calculations show that the Cys25, His159 essential ion pair is fully ionised at pH 2.99, where activity cannot be detected, but apparently an additional ionisation with a pKa of 4 is essential for activity (an electrostatic switch). Caricain (EC 3.4.22.30) wt and D158E genetic backgrounds were used to study the contribution of E50A to activity. E50 or E135 are candidates for the switch, E50A would be expected to reduce activity. However, activity increased at pH 5.0 in both backgrounds and at the pH optimum in D158E E50A but decreased slightly in the wt background. This challenges the hypothesis of an electrostatic switch.

Binding Sites↗