Protein engineering最新文献

Proposed is a method for locating functionally relevant atoms in protein structures and a representation of spatial arrangements of these atoms allowing for a flexible description of active sites in proteins. The search method is based on comparison of local structure features of proteins that share a common biochemical function. The method does not depend on overall similarity of structures and sequences of compared proteins or on previous knowledge about functionally relevant residues. The compared protein structures are condensed to a graph representation, with atoms as nodes and distances as edge labels. Protein graphs are then compared to extract all possible Common Structural Cliques. These cliques are merged to create Structural Templates: graphs that describe structural analogies between compared proteins. Structures of serine endopeptidases were compared in pairs using the presented algorithm with different geometrical parameters. Additionally, a Structural Template was extracted from the structures of aminotransferases, two different proteins that catalyze the same type of chemical reaction. The results presented show that the method works efficiently even in the case of large protein systems and allows for extraction of common structural features from proteins catalyzing a particular chemical reaction, but that evolved from different ancestors by convergent evolution.

The prediction of protein secondary structure is an important step in the prediction of protein tertiary structure. A new protein secondary structure prediction method, SVMpsi, was developed to improve the current level of prediction by incorporating new tertiary classifiers and their jury decision system, and the PSI-BLAST PSSM profiles. Additionally, efficient methods to handle unbalanced data and a new optimization strategy for maximizing the Q(3) measure were developed. The SVMpsi produces the highest published Q(3) and SOV94 scores on both the RS126 and CB513 data sets to date. For a new KP480 set, the prediction accuracy of SVMpsi was Q(3) = 78.5% and SOV94 = 82.8%. Moreover, the blind test results for 136 non-redundant protein sequences which do not contain homologues of training data sets were Q(3) = 77.2% and SOV94 = 81.8%. The SVMpsi results in CASP5 illustrate that it is another competitive method to predict protein secondary structure.

Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity. For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli. The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer). Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance. The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities. Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells. This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies.

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI >> GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.

The alpha-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis alpha-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.

Proteins are thought to undertake single substitutions, deletions and insertions to explore the fitness landscape. Nevertheless, the ways in which these different kind of mutations act together to alter a protein phenotype remain poorly described. We introduced incrementally the single substitution W290A and a 26 amino acid long insertion at the 297 location in the Nippostrongylus brasiliensis acetylcholinesterase B sequence and analysed in vitro the induced changes in the hydrolysis rate of three hemi-substrates: pirimicarb, paraoxon methyl and omethoate. The substitution decreased the hydrolysis rate of the three hemi-substrates. The insertion did not influence this kinetic alteration induced by the substitution for the former hemi-substrate, but reverted it for the two others. These results show that two different kinds of mutations can interact together to influence the direction of a protein's adaptative walk on the fitness landscape.

Winged bean chymotrypsin inhibitor (WCI) has an intruding residue Asn14 that plays a crucial role in stabilizing the reactive site loop conformation. This residue is found to be conserved in the Kunitz (STI) family of serine protease inhibitors. To understand the contribution of this scaffolding residue on the stability of the reactive site loop, it was mutated in silico to Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD) simulations were carried out on the mutants. The results of MD simulations reveal the conformational variability and range of motions possible for the reactive site loop of different mutants. The N-terminus side of the scissile bond, which is close to a beta-barrel, is conformationally less variable, while the C-terminus side, which is relatively far from any such secondary structural element, is more variable and needs stability through hydrogen-bonding interactions. The simulated structures of WCI and the mutants were docked in the peptide-binding groove of the cognate enzyme chymotrypsin and the ability to form standard hydrogen-bonding interactions at P3, P1 and P2' residues were compared. The results of the MD simulations coupled with docking studies indicate that hydrophobic residues like Leu and Val at the 14th position are disruptive for the integrity of the reactive site loop, whereas a residue like Thr, which can stabilize the C-terminus side of the scissile bond, can be predicted at this position. However, the size and charge of the Asn residue made it most suitable for the best maintenance of the integrity of the reactive site loop, explaining its conserved nature in the family.

We propose a novel method for identifying and classifying the functions of transmembrane (TM) proteins based on their TM topology [the number of TM segments (tms), the loop length and the N-terminus location]. In this method, the TM topology is expressed as a string of '0' and '1', and this is designated the binary topology pattern (BTP). We focused on TM proteins with up to 12 tms, with the exception of 1 and 9 tms, and classified them into 37 functional groups by the number of tms and the functional annotation. These grouped TM protein sequences were used to determine BTPs which are specific to the individual functional groups. Since the evaluated accuracies (sensitivity, specificity and self-consistency) of these patterns in functional identification were quite high overall, i.e. 0.940, 0.934 and 0.935, respectively, as averaged over the 37 functional groups, we confirmed that TM protein function can be identified by the number of tms and the characteristics of loop lengths, i.e. BTPs.

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.