A rigid network of long-range contacts increases thermostability in a mutant endoglucanase

Thermodynamic stability of a protein at elevated temperatures is a key factor for thermostable enzymes to catalyze their specific reactions. Yet our understanding of biological determinants of thermostability is far from complete. Many different atomistic factors have been suggested as possible means for such proteins to preserve their activity at high temperatures. Among these factors are specific local interatomic interactions or enrichment of specific amino acid types. The case of glycosyl hydrolase family endoglucanase of Trichoderma reesei defies current hypotheses for thermostability because a single mutation far from the active site (A35?V) converts this mesostable protein into a thermostable protein without significant change in the protein structure. This substantial change in enzymatic activity cannot be explained on the basis of local intramolecular interactions alone. Here we present a more global view of the induced thermostability and show that the A35?V mutation affects the underlying structural rigidity of the whole protein via a number of long-range, non-local interactions. Our analysis of this structure reveals a precisely tuned, rigid network of atomic interactions. This cooperative, allosteric effect promotes the transformation of this mesostable protein into a thermostable one.     

Mapping long-range contacts in a highly unfolded protein

Insights into the earliest events in protein folding can be obtained by analysis of the conformational propensities of unfolded or partly folded states. The structure of the acid-unfolded state of apomyoglobin has been characterized using paramagnetic spin labeling and NMR. Nitroxide side-chains, introduced by coupling to mutant cysteine residues at positions 18, 77, and 133, were used as probes of chain compaction and long-range tertiary contacts. Significant interactions are observed within and between the N and C termini, while the central region of the polypeptide chain behaves as a random polymer. Even in this highly denatured form, the protein samples transient compact states in which there are native-like contacts between the N and C-terminal regions.     

Dynamics of endoglucanase catalytic domains: implications towards thermostability

Thermostable endoglucanases play a crucial role in the production of biofuels to breakdown plant cellulose. Analyzing their structure-dynamics relationship can inform about the origins of their thermostability. Although tertiary structures of many endoglucanase proteins are available, the relationship between thermostability, structure, and dynamics is not explored fully. We have generated elastic network models for thermostable and mesostable endoglucanases with the (αβ)? fold in substrate bound and unbound states. The comparative analyses shed light on the relation between protein dynamics, thermostability, and substrate binding. We observed specific differences in the dynamic behavior of catalytic residues in slow modes: while both the nucleophile and the acid/base donor residues show positively correlated motions in the thermophile, their dynamics is uncoupled in the mesophile. Our proof-of-concept comparison study suggests that global dynamics can be harnessed to further our understanding of thermostability.     

Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum

A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Yi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86°C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85°C. The results provide valuable thermostability-related structural information on this thermophilic enzyme.     

A mutant subtilisin E with enhanced thermostability

A mutant subtilisin E with remarkably thermostability is reported. It is more active against the typical substrate s-AAPF-pna than the wild-type subtilisin E. The time required for getting 50% residual activity of Ser236Cys subtilisin E at 60 °C in aqueous solution was approximately 80 min which is 4 times longer than that of wild-type subtilisin E. Similar to the wild-type subtilisin E, the amidase activity of Ser236Cys subtilisin E is dramatically reduced in the presence of dimethylformamide (DMF).     

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.     

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.     

PROFcon: novel prediction of long-range contacts

MOTIVATION: Despite the continuing advance in the experimental determination of protein structures, the gap between the number of known protein sequences and structures continues to increase. Prediction methods can bridge this sequence-structure gap only partially. Better predictions of non-local contacts between residues could improve comparative modeling, fold recognition and could assist in the experimental structure determination. RESULTS: Here, we introduced PROFcon, a novel contact prediction method that combines information from alignments, from predictions of secondary structure and solvent accessibility, from the region between two residues and from the average properties of the entire protein. In contrast to some other methods, PROFcon predicted short and long proteins at similar levels of accuracy. As expected, PROFcon was clearly less accurate when tested on sparse evolutionary profiles, that is, on families with few homologs. Prediction accuracy was highest for proteins belonging to the SCOP alpha/beta class. PROFcon compared favorably with state-of-the-art prediction methods at the CASP6 meeting. While the performance may still be perceived as low, our method clearly pushed the mark higher. Furthermore, predictions are already accurate enough to seed predictions of global features of protein structure.     

Environment-dependent long-range structural distortion in a temperature-sensitive point mutant

Extensive environment-dependent rearrangement of the helix-turn-helix DNA recognition region and adjacent L-tryptophan binding pocket is reported in the crystal structure of dimeric E. coli trp aporepressor with point mutation Leu75Phe. In one of two subunits, the eight residues immediately C-terminal to the mutation are shifted forward in helical register by three positions, and the five following residues form an extrahelical loop accommodating the register shift. In contrast, the second subunit has wildtype-like conformation, as do both subunits in an isomorphous wildtype control structure. Treated together as an ensemble pair, the distorted and wildtype-like conformations of the mutant apoprotein agree more fully than either conformation alone with previously reported NOE measurements, and account more completely for its diverse biochemical and biophysical properties. The register-shifted segment Ile79-Ala80-Thr81-Ile82-Thr83 is helical in both conformations despite low helical propensity, suggesting an important structural role for the steric constraints imposed by β-branched residues in helical conformation.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

Overproduction of a Clostridium cellulolyticum endoglucanase by mutant strains of Escherichia coli

We have studied the expression of an endoglucanase from Clostridium cellulolyticum in mutant strains of Escherichia coli that overproduce haemolysin. When these mutants were transformed with plasmids encoding the endoglucanase, they showed a significantly enhanced endoglucanase activity, compared to transformed parental strains. Among the mutants, strain Hha-2 showed the highest production. We have identified the endoglucanase gene product synthesized in E. coli Hha-2/pBP8 and detected an increased amount of the enzyme parallel to the increase of endoglucanase activity. This was mainly localized in the periplasm and only a small percentage of it was found in the culture fluid.     

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

Background  

In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.     

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

Glycosylation increases the thermostability of human aquaporin 10 protein

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.     

A neural network based predictor of residue contacts in proteins

We describe a method based on neural networks for predicting contact maps of proteins using as input chemicophysical and evolutionary information. Neural networks are trained on a data set comprising the contact maps of 200 non-homologous proteins of well resolved three-dimensional structures. The systems learn the association rules between the covalent structure of each protein and its correspondent contact map by means of a standard back propagation algorithm. Validation of the predictor on the training set and on 408 proteins of known structure which are not homologous to those contained in the training set indicate that this method scores higher than statistical approaches previously described and based on correlated mutations and sequence information.     

A mutant gene that increases gibberellin production in brassica

A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A₃ (GA₃) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA₁ and GA₃ were estimated by gas chromatography-selected ion monitoring using [²H]GA₁, as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA₂₀ and GA₁, and the rate of GA₁₉ metabolism were simultaneously analyzed at day 7 by feeding [²H₂]GA₁₉ and measuring metabolites [²H₂]GA₂₀ and [²H₂]GA₁ and endogenous GA₂₀ and GA₁, with [²H₅]GA₂₀ and [²H₅]GA₁ as quantitative internal standards. Levels of GA₁ and GA₂₀ were 4.6- and 12.9-fold higher, respectively, and conversions to GA₂₀ and GA₁ were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA₁ biosynthesis in ein, the conversion of [³H]GA₂₀ to [³H]GA₁ was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA₁ biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A₁ and A₃. The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation.     

Altered DNA contacts made by a mutant AraC protein.

Mutant AraC proteins were selected for their ability to induce but not to repress, or their ability to repress but not to induce the araBAD operon. One such unusual mutant is able to bind to the araI site with an affinity only two to three-fold weaker than the wild type AraC protein, but the mutant protein was shown, both in crude extracts and when purified, to contact only two of the three major groove regions of the DNA that are contacted by the wild type protein.     

Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells

Protein denaturation resulting from temperatures between 42.0 degrees C and 50 degrees C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthermic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42 degrees C and 52 degrees C in control cells by increasing their transition (denaturation) temperature by an average of 1.3 degrees C. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp.

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.