Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

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.     

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Dihydrofolate reductase mutants with amino acid replacementsin the active center (Thr35 Asp mutant, Arg57 His mutant andthe mutant with triple replacement Thr35 Asp, Asn37 Ser, Arg57 His) were obtained by site-directed mutagenesis. The stabilizationeffect of trimethoprim and NADP·H on the protein tertiarystructure in vitro has been investigated. In the case of mutantswith a ‘weak’ tertiary structure (Thr35 Asp35 andthe triple mutant) the separate addition of ligands does notaffect their stability. The simultaneous addition of these ligandsto Thr35 Asp35 and the triple mutant leads to the large increasein their stability. A distinct correlation was found betweenthe in vitro studied stability of the mutant proteins to theurea- or heat-induced denaturation and the level of proteolyticdegradation of these mutants previously observed in vivo.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of {alpha}-helices

The 247–260 and 289–299 -helices of Bacillus subtilisneutral protease have a lysine in their N-terminal turn. Theselysines were replaced by Ser or Asp in order to improve electrostaticinteractions with the -helix dipole. After replacing Lys bySer at positions 249 or 290, the thermostability of the enzymewas increased by 0.3 and 1.0°C, respectively. The Asp249and Asp290 mutants exhibited a stabilization of 0.6 and 1.2°C,respectively. The results show the feasibility of stabilizingenzymes by introducing favourable residues at the end of -helices.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

The differences in conformation between {alpha}-human atrial natriuretic polypeptide, {alpha}-hANP, and its derivative, Met(O)-{alpha}-hANP, in solution

The differences in conformation between -human atrial natriureticpolypeptide (-hANP) and its inactive analog, Met(O)--hANP, havebeen analyzed by nuclear magnetic resonance spectroscopy. Allproton resonances for both peptides were assigned by means ofthe sequential assignment procedure. The three-dimensional structureof -hANP in solution had previously been determined by distancegeometry calculation using distance constraints derived fromnuclear Overhauser effects (NOEs). Here, the three-dimensionalstructure of Met(O)--hANP was determined. The conformationaldifferences between these two molecules were as follows: threesegments of -hANP, Serl–Cys7, Arg11–Ala17 and Gln18–Tyr28,have some ordered structures. In Met(O)--hANP the Gln18-Tyr28region has a similar conformation, while the remaining two regionsdo not have the ordered structure found in -hANP. It is suggestedthat the conserved conformation of the Gln18–Tyr28 regionis required for binding to the ANP receptor and that the slightbiological activity of Met(O)-a-hANP is due to loss of the orderedstructures evoked in the Serl–Cys7 and Arg11–Ala17regions of -hANP.     

Synthesis and characterization of a recombinant fragment of human {alpha}-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human -fetoprotein amino acid sequence38–119 was synthesized and cloned in a bacterial expressionvector. The -fetoprotein sequence was selected as the leasthomologous to albumin, since the two proteins have an overallamino acid identity of %. A chimeric protein was obtained whichwas purified by preparative electrophoresis and characterizedin its primary structure by fast atom bombardment mass spectrometry.About 70% of the -fetoprotein sequence was physically mappedand found to correspond to the amino acids encoded in the syntheticgene. The use of this recombinant protein allowed the selectionof monoclonal antibodies recognizing both the recombinant fragmentand native -fetoprotein. These antibodies should allow the developmentof an immunoassay for -fetoprotein with absolute selectivityversus albumin. This might result in more sensitive clinicaldeterminations, avoiding the possibility of cross-reactions.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide -calcitonin gene-related peptide(-CGRP). Both amylin and -CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an -helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.     

{alpha}-Helix folding by Monte Carlo simulated annealing in isolated C-peptide of ribonuclease A

Conformation of the C-peptide fragment of RNase A is calculatedby Monte Carlo simulated annealing. We adopt the total potentialenergy as given by the sum of generic interatomic energies whoseparameters are determined separately for each amino acid withoutreferring to the empirical structure of the C-peptide. The simulationis carried out in a completely unrestricted way without imposingany weight towards given final destinations. Starting from completelyrandom initial conformations and minimizing the total potentialenergy with respect to main-chain dihedral angles and side-chaintorsion angles, we have obtained partial -helix structure witha high probability ({small tilde}40%). The energetically mostfavourable structure exhibits a 2.5-turn -helix at the locationidentical with that of the 3-turn -helix in the native enzymemolecule. Classification of conformations obtained in the simulationinto clusters of similar structure shows that our simulationindeed predicts the -helix structure for the isolated C-peptidewith specific charged residues. The results of simulation withvarious amino acid substitutions are also found to be consistentwith the experimental implication for the importance of intramolecularionic interactions for -helix stability for this peptide.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Engineering of protein epitopes: a single deletion in a snake toxin generates full binding capacity to a previously unrecognized antibody

Structural features associated with the ability of a monoclonalantibody (mAb) to discriminate between protein variants areidentified and engineered. The variants are the curaremimetictoxin from Naja nigricollis and erabutoxin a or b from Laticaudasemifasciata which differ from each other by 16 substitutionsand one insertion. The neutralizing mAb M1 recognizes with highaffinity a topographical epitope on the surface of toxin , butfails to recognize the erabutoxins although they possess mostof the residues forming the presumed epitope. Examinations ofthe toxin and erabutoxin 3-D structures and molecular dynamicssimulations reveal several differences between the variants.In particular, the region involving the ß-turn 17–24is organized differently. Analysis of the differences foundin this region suggests that the insertion (or deletion) atposition 18 of the variant amino add sequences is particularlyimportant in determining the differential cross-reactivity.To test this proposal, residue 18 was deleted in one erabutoxinusing sitedirected mutagenesis, and the biological propertiesof the resulting mutant were examined. We found that full antigenicitywas restored in the previously unrecognized variant. The implicationsof this finding are discussed.     

Construction of multiple copy of {alpha}-domain gene fragment of human liver metallothionein IA in tandem arrays and its expression in transgenic tobacco plants

Metallothioneins (MT) are low molecular weight, cysteinerich,metal-binding proteins. An MT molecule contains two domainswhich appear to act independently—an -domain, which ischaracterized by cadmium-binding, and a ß-domain,which binds preferentially to copper. Based on this conception,DNA duplex encoding the -domain (106 bp) of human MT-I_A wasconstructed from a chemically-synthesized oligomer by repairsynthesis and enzymatic ligation and cloned into pUC19. Thegenes cloned were sequenced and found to be in the correct orderas designed. Synthetic directional adapters were attached tothe terminals of the -domain gene fragment of human MT-I_A toestablish complete control over fragment orientation duringligation. The use of these directional adapters thereby ensuredthe production of multiple copies of the -domain in tandem arrays.The successive -domains were linked by a peptide linker consistingof 10 residues. A chimeric gene containing 12 cloned tandemlyrepeated copies of the 106 bp -domain DNA was introduced intotobacco cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens.A total of 10 different transgenic tobacco plants were generated,of which two showed root and shoot growth unaffected by up to200 mg/l kanamycin and 100 µM cadmium, whereas root growthof control plants was severely inhibited and leaf chlorosisdeveloped on media containing only 10 µM cadmium     

Independent self-assembly of cadmium-binding {alpha}-fragment of metallothionein in Escherichia coli without participation of {beta}-fragment

We examined the independent self-assembly of the - and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed -fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The -fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed.     

Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys⁴⁷ ), was found to be a competitiveinhibitor of human -thrombin with respect to peptidyl p-Miitroanilidesubstrates. These results contrast with those of Degryse andcoworkers that suggest that recombinant hirudin variant-2(Lys⁴⁷)inhibited thrombin by a non-competitive mechanism [Degryse etal. (1989) Protein Engng, 2, 459–465], -Thrombin, whichcan arise from -thrombin by autolysis, was shown to have anaffinity for recombinant hirudin variant-2(Lys⁴⁷) that was fourorders of magnitude lower than that of -thrombin. It was demonstratedthat the apparent noncompetitive mechanism observed previouslywas probably caused by a contamination of the thrombin preparationby -thrombin. Comparison of the inhibition of -thrombin by recombinanthirudins variant-2(Lys⁴⁷) and variant-1, which differ from oneanother in eight out of 65 amino acids, indicated that the twovariants have essentially the same kinetic parameters.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Expression of recombinant {alpha}Ains-crystallin and not {alpha}A-crystallin inhibits bacterial growth

A-Crystallin and A^ins-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the ‘insert’ exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and A^ins-crystallinare functionally equivalent and that the presence of the ‘insert’peptide in A^Ins-crystallin is inconsequential. We report herethat the independent expression of recombinant A^Ins-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof A^Ins-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and A^Ins-crystallin differby a peptide of 23 amino acids, these data suggest that the‘insert peptide’ in A^Ins-crystallin imparts propertieson this protein that are different from A-crystallin.     

Stability, activity and flexibility in {alpha}-lactalbumin

-Lactalbumins and the type-c lysozymes are homologues with similarfolds that differ in function and stability. To determine ifthe lower stability of -lactalbumin results from specific substitutionsrequired for its adaptation to a new function, the effects oflysozyme-based and other substitutions on thermal stabilitywere determined. Unblocking the upper cleft in -lactalbuminby replacing Tyr103 with Ala, perturbs stability and structurebut Pro, which also generates an open cleft, is compatible withnormal structure and activity. These effects appear to reflectalternative enthalpic and entropic forms of structural stabilizationby Tyr and Pro. Of 23 mutations, only three, which involve substitutionsfor residues in flexible substructures adjacent to the functionalsite, increase stability. Two are lysozyme-based substitutionsfor Leu110, a component of a region with alternative helix andloop conformations, and one is Asn for Lys114, a residue whosemicroenvironment changes when -lactalbumin interacts with itstarget enzyme. While all substitutions for Leu110 perturb activity,a Lys114 to Asn mutation increases T_m by more than 10°Cand reduces activity, but two other destabilizing substitutionsdo not affect activity. It is proposed that increased stabilityand reduced activity in Lys114Asn result from reduced flexibilityin the functional site of -lactalbumin.