Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins

The biological activity of some proteins is known to be sensitiveto oxidative damage caused by a variety of oxidants. The modelprotein staphylococcal nuclease was used to explore the effecton protein structural stability of oxidizing methionine to thesulfoxide form. These effects were compared with the effectsof substituting methionines with isoleucine and leucine, a potentialstrategy for stabilizing proteins against oxidative damage.Wild-type nuclease and various mutants were oxidized with hydrogenperoxide. Stabilities of both oxidized and unoxidized proteinswere determined by guanidine hydrochloride denaturation. Oxidationdestabilized the wild-type protein by over 4 kcal/mol. Thislarge loss of stability supports the idea that in some casesloss of biological activity is linked to disruption of the proteinnative state. Comparison of mutant protein's stability lossesupon oxidation showed that methionines 65 and 98 had a muchgreater destabilizing effect when oxidized than methionines26 or 32. While substitution of methionine 98 carried as greatan energetic penalty as oxidation, substitution at position65 was less disruptive than oxidation. Thus a simple substitutionmutagenesis strategy to protect a protein against oxidativedestabilization is practical for some methionine residues.     

Limited hydrolysis of soy proteins with endo- and exoproteases

Changes in the native state and functional properties of soy protein achieved by limited proteolysis of soy flour were investigated. Different enzyme-to-substrate ratios (E/S) were used to obtain low (3–5%) and medium (5–10%) degrees of hydrolysis (DH). Six protease preparations (three with predominately exopeptidase activities and three with predominately endopeptidase activities) were evaluated, and their effects on solubility, emulsification capacity, SDS-PAGE profiles, and denaturation enthalpies were characterized. Endoproteases (Multifect^® Neutral, Protex^? 6L, and Multifect^® P-3000) and exoproteases (Fungal Protease Concentrate, Experimental Fungal Protease #1, and Experimental Fungal Protease #2) yielded similar increases in soy protein solubility. The modifications to the soy peptide profile were similar for the three exoprotease mixtures at a 1% E/S ratio, whereas the extent of hydrolysis with Protex^? 6L was more pronounced than with the two other endoproteases (Multifect^® Neutral and Multifect^® P-3000). The emulsification capacity of protease-modified soy flour declined regardless of DH and enzyme type (exo- or endoprotease). After hydrolysis to >4% DH, denaturation enthalpies of glycinin and β-conglycinin decreased significantly, whereas hydrolysis to lower DH did not affect these values.     

Development of antiviral fusion inhibitors: short modified peptides derived from the transmembrane glycoprotein of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) is a naturally occurring pathogen that causes an AIDS-like syndrome in domestic cats and is a valuable model system by which criteria for antiviral vaccines and drugs development can be tested. The cell-entry step of the lentivirus life cycle is regarded as a promising target for the development of new generation inhibitors. We have previously described potent in vitro anti-FIV activity associated with a synthetic octapeptide, termed C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), containing the Trp-rich motif of FIV transmembrane glycoprotein, which shares a common structural framework with the corresponding molecule of HIV and appears to play a similar role in cell entry. In this report, in an attempt to develop simpler potential fusion inhibitors to be tested in vivo, we describe further studies focused on synthetic peptide analogues of C8. Since C8 inhibitory activity is dependent upon the Trp motif, we systematically replaced these residues with bulky and/or aromatic natural and unnatural amino acids, in order to develop a rational structure-activity relationship. Furthermore, the amino acids located between the Trp residues, which are not crucial for inhibitory activity, were replaced by simple alkyl spacers of appropriate length. Design, NMR structural analysis, in vitro anti-FIV activity in lymphoid cell cultures, and serum stability of these new analogues are reported. The final results indicate that a simpler hexapeptide (Ac-Nal2-Ape-Nal2-Ape-Nal2-Ile-NH2; Nal2 = 3-naphthalen-2-yl-L-alanine, Ape = 5-aminopentanoic acid), almost entirely made up of unnatural amino acid residues, has markedly increased enzymatic stability, while maintaining strong antiviral potency in vitro.     

Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori

The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed. First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method. To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay. Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity. The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed.      

Isolation of an active peptide fragment from human serum albumin and its synergism with α-tocopherol

An active peptide was isolated from hydrolysates of human serum albumin. This peptide was initially isolated by gel permeation chromatography and subsequent reversed-phase high-performance liquid chromatography. This active peptide, composed of 10 amino acid residues, was further hydrolyzed with a lysyl endopeptidase to give two peptide fragments. Only one fragment, identified as the tetrapeptide Leu-Gln-His-Lys, was found to have activity comparable to the original peptide and corresponded to the amino acid residues 103–106 of human serum albumin. Among these four amino acid residues, the His-Lys sequence seemed to be important in the occurrence of potent activity by comparison of the structural similarity with another active tetrapeptide, Asp-Thr-His-Lys, which had been previously isolated from bovine serum albumin hydrolysates. In addition, the active fragment showed potent synergism by preventing consumption of α-tocopherol during the autoxidation of linoleic acid.     

Activity- and Enrichment-Based Metaproteomics Insights into Active Urease from the Rumen Microbiota of Cattle

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.     

Isolation of an active peptide fragment from human serum albumin and its synergism with α-tocopherol

An active peptide was isolated from hydrolysates of human serum albumin. This peptide was initially isolated by gel permeation chromatography and subsequent reversed-phase high-performance liquid chromatography. This active peptide, composed of 10 amino acid residues, was further hydrolyzed with a lysyl endopeptidase to give two peptide fragments. Only one fragment, identified as the tetrapeptide Leu-Gln-His-Lys, was found to have activity comparable to the original peptide and corresponded to the amino acid residues 103–106 of human serum albumin. Among these four amino acid residues, the His-Lys sequence seemed to be important in the occurrence of potent activity by comparison of the structural similarity with another active tetrapeptide, Asp-Thr-His-Lys, which had been previously isolated from bovine serum albumin hydrolysates. In addition, the active fragment showed potent synergism by preventing consumption of α-tocopherol during the autoxidation of linoleic acid.     

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.     

Effect on inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficientlyinhibits a bacterial serine protease, subtilisin BPN'. We recentlydemonstrated that functional change in SSI was possible simplyby replacing the amino acid residue at the reactive P1 site(methionine 73) of SSI. The present paper reports the additionaleffect of replacing methionine 70 at the P4 site of SSI(Lys73)on inhibitory activity toward two types of serine proteases,trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversionof methionine 70 at the P4 site of SSI(Lys73) to glycine oralanine resulted in increased inhibitory activity toward trypsinand lysyl endopeptidase, while replacement with phenylalanineweakened the inhibitory activity toward trypsin. This suggeststhat steric hindrance at the P4 site of SSI(Lys73) is an obstaclefor its binding with trypsin. In contrast, the same P4 replacementshad hardly any effect on inhibitory activity toward subtilisinBPN'. Thus the subsite structure of subtilisin BPN' is tolerantto these replacements. This contrast in the effect of P4 substitutionmight be due to the differences in the S4 subsite structuresbetween the trypsin-like and the subtilisin-like proteases.These findings demonstrate the importance of considering structuralcomplementarity, not only at the main reactive site but alsoat subsites of a protease, when designing stronger inhibitors.     

Insights into the Structure of Rubisco from Dinoflagellates-In Silico Studies

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere’s productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme’s activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.     

Cytochrome P450 OxyBtei Catalyzes the First Phenolic Coupling Step in Teicoplanin Biosynthesis

Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as “Oxy enzymes” play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non‐ribosomal antibiotic precursor peptide while it remains bound to the non‐ribosomal peptide synthetase (NRPS); this crosslinking secures the three‐dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyB_tei, the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyB_tei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS‐bound peptide substrates. However, OxyB_tei displays a significantly altered activity spectrum against peptide substrates compared to its well‐studied vancomycin homologue.     

Dynamic Coupling of Tyrosine 185 with the Bacteriorhodopsin Photocycle,as Revealed by Chemical Shift Assisted AF-QM/MM Calculations and Molecular Dynamic Simulations

Aromatic residues are highly conserved in microbial photoreceptors and play crucial roles in the dynamic regulation of receptor functions. However, little is known about the dynamic mechanism of the functional role of those highly conserved aromatic residues during the receptor photocycle. Tyrosine 185 (Y185) is one of the highly conserved aromatic residues within the retinal binding pocket of bacteriorhodopsin (bR). In this study, we explored the molecular mechanism of its dynamic coupling with the bR photocycle by automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) calculations and molecular dynamic (MD) simulations based on chemical shifts obtained by 2D solid-state NMR correlation experiments. We observed that Y185 plays a significant role in regulating the retinal cis–trans thermal equilibrium, stabilizing the pentagonal H-bond network, participating in the orientation switch of Schiff Base (SB) nitrogen, and opening the F42 gate by interacting with the retinal and several key residues along the proton translocation channel. Our findings provide a detailed molecular mechanism of the dynamic couplings of Y185 and the bR photocycle from a structural perspective. The method used in this paper may be applied to the study of other microbial photoreceptors.     

Bioorthogonal Metalloporphyrin-Catalyzed Selective Methionine Alkylation in the Lanthipeptide Nisin

Bioorthogonal catalytic modification of ribosomally synthesized and post-translationally modified peptides (RiPPs) is a promising approach to obtaining novel antimicrobial peptides with improved properties and/or activities. Here, we present the serendipitous discovery of a selective and rapid method for the alkylation of methionines in the lanthipeptide nisin. Using carbenes, formed from water-soluble metalloporphyrins and diazoacetates, methionines are alkylated to obtain sulfonium ions. The formed sulfonium ions are stable, but can be further reacted to obtain functionalized methionine analogues, expanding the toolbox of chemical posttranslational modification even further.     

Cellulolytic properties of Chaetomium crispatum

The cellulolytic properties of a Chaetomium crispatum strain were investigated. The cellulolytic enzyme complex i.e.: exo-1,4-β-glucosidase (EC 3.2.1.74); endo-1,4-β-glucanase (EC 3.2.1.4.) and β-glucosidase or cellobiase (EC 3.2.1.21) displayed optimal activity at pH 5.0 and 25°C. Although carboxymethyl-celluloses are the usual pseudo-substrates for this enzyme complex, those with a high degree of substitution gave rise to poor growth and low cellulase activity. Insoluble crude cellulosics such as newsprint, recycled paper, rice and flax straw were substantially solubilised at 28°C within 3–5 days of fermentation. A study of the cellulase-complex formation during the growth cycle revealed that β-glucosidase was produced mainly intracellularly in the early exponential phase, while the overall exo-1,4-β-glucosidase and endo-1,4-β-glucanase formation gradually increased during the total fermentation cycle. The mycelial protein of Chaetomium crispatum grown on crude cellulosics displayed a favourable amino acid pattern, indicating its potential value as a source of single cell protein (SCP).     

Dissecting key residues in folding and stability of the bacterial immunity protein 7

The small four-helix immunity protein, Im7, has previously been shown to fold via a compact intermediate containing three of the four native helices. The short, six-residue helix III only docks onto the developing Im7 structure after the rate-limiting second transition state has been traversed. Previous work demonstrated that mutation of the helix III sequence can be used to trap the protein in the on-pathway intermediate ensemble at equilibrium. Here the role played by individual residues in the native helix III sequence in locking Im7 into a stable native structure is further examined. This work commenced with an Im7 sequence trapped in the partially folded state by substitution of the six residues in helix III with a polyglycine sequence. Biophysical analysis of variants in which individual residues from the native helix III sequence, and combinations of these residues, were introduced into this background demonstrated a critical requirement for three residues, Leu 53, Ile 54 and Tyr 55, to lock Im7 into its unique native structure. The results demonstrate a stringent constraint on the evolution of the Im7 helix III sequence rationalizing its high-sequence identity in the fold family. Thus, Leu 53 and Ile 54 provide crucial stabilizing interactions in the hydrophobic core of native Im7, while Tyr 55 is required for both stability and function. In contrast, Tyr 56 is critical for colicin binding and has no role in maintaining a stable native fold.     

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase

The large form of glucoamylase (GAI) from Aspergillus awamori(EC 3.2.1.3 [EC] ) binds strongly to native granular starch, whereasa truncated form (GAII) which lacks 103 C-terminal residues,does not. This C-terminal region, conserved among fungal glucoamylasesand other starch-degrading enzymes, is part of an independentstarch-binding domain (SBD). To investigate the SBD boundariesand the function of conserved residues in two putative substrate-bindingsites, five gluco-amylase mutants were constructed with extensivedeletions in this region for expression in Saccharomyces cerevisiae.Progressive loss of both starch-binding and starch-hydrolyticactivity occurred upon removal of eight and 25 C-terminal aminoacid residues, or 21 and 52 residues close to the N-terminus,confirming the requirement for the entire region in formationof a functional SBD. C-terminal deletions strongly impairedSBD function, suggesting a more important role for one of theputative binding sites. A GAII phenocopy showed a nearly completeloss of starch-binding and starch-hydrolytic activity. The deletionsdid not affect enzyme activity on soluble starch or thermo-stabilityof the enzyme, confirming the independence of the catalyticdomain from the SBD.     

Structure-activity studies on the corticotropin releasing factor antagonist astressin, leading to a minimal sequence necessary for antagonistic activity

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.     

Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease

On the basis of the homology with the Bacillus thermoproteolyticuszinc endopeptidase thermotysin, we hypothesized that Glu-143and His-231 are the key residues for the catalytic activityof the Bacillus subtilis neutral protease. To test this possibilityby site-directed mutagenesis, we substituted these two residueswith Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively.All these substitutions dramatically affected the amount ofsecreted mutant proteins, as determined by immunological methods,and their catalytic activities. No appreciable secretion wasobserved with the three Glu mutants Trp, Ser and Arg, whereasthe Glu–Ala mutant enzyme was secreted at a level of afew hundred micrograms per litre of culture. The His mutantswere all secreted at higher levels (in the order of a few milligramsper litre) and their residual catalytic activity could be determinedusing Z-Ala-Leu-Ala as substrate. Our results confirm the keyrole played by Glu-143 and His-231 in catalysis and moreoversuggest the existence of a relationship between the catalyticactivity of the enzyme and the extent of its secretion. In thiscontext, we present data suggesting an autoproteolytic mechanismof cleavage of the precursor form of the enzyme, analogous tothe one previously reported for the B.subtilis subtilisin.     

Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group

Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine-specific reagent 4,5-dimethoxy-2-nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active-site Cys141 with Ser resulted in an approximately 100-fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near-ultraviolet light (320-400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.