Generation of analogs having potent antimicrobial and hemolytic activities with minimal changes from an inactive 16-residue peptide corresponding to the helical region of Staphylococcus aureus {delta}-toxin

The -toxin is a 26-residue peptide from Staphylococcus aureuswith the sequence formyl-MAQDIISTIGDLVKWIIDTVNKFTKK. NMR studiesindicate that the segment IISTIGDLVKWIIDTV occurs in an -helicalconformationin the toxin. A synthetic peptide correspondingto this segment, although helical, did not exhibit hemolyticactivity. Since charged residues like D and K are likely tomodulate cytolytic activity, analogs of the 16-residue peptidewere synthesized where D was systematically replaced by K. Analogsin which the first D and both Ds were replaced by K showed potentantimicrobial and hemolytic activities. The analog in whichthe second D was replaced by K was relatively less active. However,all the peptides showed an -helical structure with similar helicalcontent. The activities of the peptides were found to correlatedirectly with their ability to permeabilize model membranes.Thus, by minimal judicious replacement of charged amino acids,it should be possible to generate cytolytic peptides from shortsegments of peptide toxins.     

家蝇抗菌肽抑菌活性及其稳定性研究

通过体外抑菌实验,采用纸片扩散法对家蝇抗菌肽的抑菌性进行定性研究。试验用菌有金黄色葡萄球菌、大肠杆菌、绿脓杆菌、白色念珠菌和黑曲霉菌。在对家蝇抗菌肽抗菌谱研究的基础上,进一步用肉汤稀释法（试管法）定量测定其最低抑菌浓度（MIC）,并对其稳定性进行研究。结果表明,家蝇抗菌肽对金黄色葡萄球菌、绿脓杆菌和大肠杆菌3种细菌有较好的抑制作用,而对白色念珠菌和黑曲霉没有明显的抑制作用,对金黄色葡萄球菌、绿脓杆菌和大肠杆菌最低抑菌浓度（MIC）分别为0.156 mg/mL、0.312 5 mg/mL和6.25 mg/mL。另外,对其稳定性进行讨论,结果表明,家蝇抗菌肽的稳定性良好。     

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.     

Modulation of the enzymatic activity of papain by interdomain residues remote from the active site

The two main catalytic residues Cys25 and Hisl59 of the monomericcysteine protease papain are located on different walls of acleft formed by two domains. This topology suggests a possiblerelationship between relative domain organization and catalyticmechanism. The effect on enzymatic parameters of structuralmodifications at various locations of the twodomain interfaceof papain was examined by individual or double replacementsby Ala of pairs of interacting residues. Most modificationshad no effect on enzyme activity. However, the enzyme's substrateturnover (k_cat) decreased following simultaneous alterationof the two most conserved residues, forming an apolar contactlocated 15 Å away from the active site. The pH activityprofile of the double mutant was unchanged, indicating a conservedionization state of the active site thiolate-imidazolium ionpair. This state is strongly dependent on the distance separatingthe two residues, thus suggesting that the active site geometryhas not been significantly altered. Efficient enzymatic activityin papain requires more than a correct active site geometryand is influenced by domain packing properties in a region remotefrom the active site.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala

A pathological variant of human phosphoglycerate kinase, phosphoglyceratekinase-Uppsala, associated with chronic nonspherocytic hemolyticanemia has been found to differ from the normal enzyme by substitutionof an arginine at position 206 (corresponding to position 203in yeast) by a proline. In order to understand the structuraland functional consequences of this mutation, the correspondingmutant in yeast phosphoglycerate kinase was constructed. Thethree-dimensional structure of this mutant was resolved at 2.9Å. Although the overall structure is not modified, smalllocal changes were observed. The kinetic parameters of the mutantwere not found to be greatly affected, the catalytic constantbeing lowered by only 10–20%. The most significant differencewhen compared with the wild-type enzyme is a decrease in stabilityby about 3 kcal/mol. The physiological implications of thisinstability are discussed.     

Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein

We have constructed three different plasmids containing partsof the human growth hormone gene using chemically synthesizedoligomers and cloned them for the purpose of expressing themin Escherichia coli. AB, B and BC gene segments correspondingto ABhGH (residue 1–138), BhGH (residue 44–138)and BChGH (residue 44–192) were placed under the controlof a tryptophan promoter in the expression vector. Upon inductionwith 3-indolylacrylic acid, ABhGH accumulated in cells but theBhGH and BChGH segments were not detected appreciably. Northernblotting analysis showed that the amount of mRNA transcribedfrom the AB gene segment was about ten-fold higher than thatfrom the B or BC gene segment. ABhGH was found to have insulin-likegrowth factor I (IGF-I) activity, which could be explained bythe hydrophilicity curves of these proteins.     

Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential

Cys320 of clostridial glutamate dehydrogenase, a residue closeto the coenzyme binding sitehas been replaced by serine.Themutant enzyme was successfully overproduced and purified byusing the normal protocol for the wild-type enzyme and alsobehaved indistinguishably from wild-type enzyme on native andSDS-PAGE. The specific activity was significantly enhanced inassays at both pH 7 (+90%) and pH 8 (+38%). Detailed initial-ratekinetics revealed that at pH 7 this increase was mainly attributableto a higher maximum rate, since the K_m values for both substrateswere marginally increased. In the mutant enzyme the inactivatingreaction with DTNB that characterizes the wild-type enzyme iscompletely eliminated. This proves that inactivation of thewild-type enzyme is due to modification of Cys320, that neverthelessCys320 is not strictly essential for catalytic activity andthat the remaining cysteine residue at position 144 is inaccessibleto DTNB. Provision of an engineered subunit with a correct nativestructure but with its DTNB titre decreased from 1 to 0 mol/molnow offers a valuable tool for counting subunits in hybrid oligomers     

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli

Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.     

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody

We have been interested for some time in establishing a strategyfor deriving lead compounds from macromolecule ligands suchas minibody variants. A minibody is a minimized antibody variabledomain whose two loops are amenable to combinatorial mutagenesis.This approach can be especially useful when dealing with `difficult'targets. One such target is the NS3 protease of hepatitis Cvirus (HCV), a human pathogen that is believed to infect about100 million individuals worldwide and for which an effectivetherapy is not yet available. Based on known inhibitor specificity(residues P6-P1) of NS3 protease, we screened a number of minibodiesfrom our collection and we were able to identify a competitiveinhibitor of this enzyme. We thus validated an aspect of recognitionby HCV NS3 protease, namely that an acid anchor is necessaryfor inhibitor activity. In addition, the characterization ofthe minibody inhibitor led to the synthesis of a constrainedhexapeptide mimicking the bioactive loop of the parent macromolecule.The cyclic peptide is a lead compound prone to rapid optimizationthrough solid phase combinatorial chemistry. We therefore confirmedthat the potential of turning a protein ligand into a low molecularweight active compound for lead discovery is achievable andcan complement more traditional drug discovery approaches.     

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin

A synthetic gene based on the published amino acid sequencefor Clostridium pasteurianum rubredoxin was constructed, clonedin Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promotersystem in E.coli HMS273. UV/visible spectroseopy and metal analysesindicated that the as–isolated synthetic gene productis a mixture of holo–(i.e. iron–containing) rubredoxinand zinc–substituted rubredoxin, with the latter amountingto {small tilde} 70% of the total rubredoxin. Hie UV/visibleabsorption and resonance Raman spectra of the cloned holorubredoxinare characteristic of the native rubredoxin–type ironsite. N–terminal amino acid sequencing suggests that thegene product consists of at least three polypeptide specieswith the initial sequences (approximate relative abundances):Met–Met–Lys–... (63%), blocked (30%) and Met–Lys–...(7%). The blocked portion presumably consists of a mixture ofnMet–Met–Lys–... and nMet-Lys–..., wherenMet represents an amino–blocked methionine residue.     

Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins

Using the techniques of genetic engineering and the principlesof protein de novo design, we have developed a unique affinitymatrix protein tag system as a rapid, convenient and sensitivemethod to detect, purify and characterize newly expressed recombinantpeptides or proteins from cell extracts. The method utilizestwo de novo-designed linear peptide sequences that can selectivelydimerize to form the stable protein motif, the two-stranded-helical coiled-coil. In this method, a recombinant bacterialexpression vector pRLDE has been engineered so that one of thedimerization strands (E-coil) is expressed as a C-terminal fusiontag on newly expressed peptides or proteins, while the other(K-coil) is either biotin-labeled for detection in a Westernblot-type format or immobilized on an insoluble silica supportfor selective dimerization affinity chromatography. Recombinantlyexpressed peptides from Escherichia coli containing the dimerizationtag have been produced, detected and purified using this method.The recombinant peptides were easily and clearly identifiedusing the biotin-labeled coil, while the single-step affinitypurification results indicated the purity of the affinity purifiedexpressed peptides to be >95%, as assessed by reversed-phasechromatography. The stability of the dimerization domain alsoallows for the purified peptide to be left attached to the matrix,thus creating a new peptide-bound column that can be used tostudy peptide–protein or peptide–ligand interactions.Therefore this system offers a new alternative to existing peptideor protein fusion tags and demonstrates the utility of a denovo-designed system.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Combination of allyl protection and HYCRAMTM-linker technology for the synthesis of peptides with problematical amino acids and sequences

Analogues of both the nonapeptides, bradykinin and bradykinin potentiating nonapeptide BPP_9α, were synthesized using HYCRAM^TM-technique. The bradykinin analogues were assembled by the Boc-, Ddz- and Fmoc-strategy starting with Boc-Arg(Aloc)₂-OCr–OH, Ddz-Arg(Mtr)-OCr–OH and Fmoc–Arg(Mtr)-OCr–OH. While Boc- and Ddz-strategy provide peptides in good yield and purity, the Fmoc-strategy leads to a loss of peptide from resin. For simultaneous cleavage from HYCRAM^TM-resin and removal of Aloc-side chain protection optimized conditions for catalytic cleavage with Pd° were developed. As shown by the synthesis of BPP_9αanalogues the HYCRAM^TM-linker and the chlorotrityl resin allow the assembly of peptides with the C-terminal sequence Pro-Pro by preventing dioxopiperazine formation. Since the BPP_9α sequence contains the tripeptide Trp-X-Arg an intramolecular migration of the N^G-protecting group to the indole ring under conditions used for its removal had to be avoided. By the use of HYCRAM^TM-linker in combination with Aloc protection for the guanidino group and Ddz for N^αno modification of Trp occurred. HYCRAM^TM-technology in combination with Boc-, Ddz- or Aloc/All-protecting groups facilitates the synthesis of peptides with such very labile amino acids like cis-4-hydroxyproline.     

Applications of circular dichroism for structural analysis of gelatin and antimicrobial peptides

Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW)(4) adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications.     

Knowledge based modelling of homologous proteins, part I: three-dimensional frameworks derived from the simultaneous superposition of multiple structures

An approach is described for modelling the three-dimensionalstructure of a protein from the tertiary structures of severalhomologous proteins that have been determined by X-ray analysis.A method is developed for the simultaneous superposition ofseveral protein molecules and for the calculation of an ‘averagestructure’ or ‘framework’. Investigation ofthe convergence properties of this method, in the case of bothweighted and unweighted least squares, demonstrates that bothgive a unique answer and the latter is robust for an homologousfamily of proteins. Multi-dimensional scaling is used to subgroupthe proteins with respect to structural homology. The frameworkcalculated on the basis of the family of homologous proteins,or of an appropriate subgroup, is used to align fragments ofthe known protein structures of high sequence homology withthe unknown. This alignment provides a basis for model buildingthe tertiary structure. Different techniques for using the frameworkto model the mainchain of various globins and an immunoglobulindomain in the structurally conserved regions are in vestigated.     

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent K_M for arachidonicacid and a minor(3-fold) change in its V_max. For linoleic acid,the variant has no change in its K_M and a 10-fold reductionin its V_max, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation.