High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.     

Expression of goat {alpha}-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat -lactalbumin and the3'-non-coding region was fused to cDNA of the N-terminal halfof porcine adenylate kinase which had been placed under thecontrol of the tac promoter in an expression vector in Escherichiacoli. In addition, a methionine codon was inserted between thetwo cDNAs. When the plasmid carried the full-length 3'-non-codingregion, little accumulation of the fused protein was observed.However, the deletion of two-thirds of the 3'-non-coding regionproduced significant expression of the fused protein in E.colistrain JM105. Since goat -lactalbumin contains no methionineresidue, the mature goat -lactalbumin was isolated by CNBr digestionof the fused insoluble protein and refolded using thioredoxin.The homogeneous and biologically active goat -lactalbumin waspurified by Ca²⁺ ion-dependent hydrophobic chromatography.     

Structure of {alpha}-{alpha}-hairpins with short connections

This paper presents the results of detailed stereochemical analysisof structures and sequences of --hairpins with short connections.It is shown that --hairpins of each given type have very similarpatterns of hydrophobic, hydrophilic and glycine residues intheir amino acid sequences. These results can be used in theprediction of --hairpin conformation as well as in protein designand engineering.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different cIts phages carryingtsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations werecloned in plasmid. The positions of these mutations and theresulting changes of amino acids in the repressor were determinedby DNA sequencing. The first four mutations mapping in the N-terminaldomain show the following changes: I21S, G53S, A62T and V73A,respectively. Of the three remaining mutations mapping in theC-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutionsrespectively, while the mutant cItsU51 gene carries F141I andP153L substitutions. Among these ts repressors, CIts2 havingthe charge-reversal change K224E was overexpressed from tacpromoter in a plasmid and purified, and its structure and functionwere studied. Operator-binding studies suggest that the ts2repressor is somewhat defective in monomer–dimer equilibriumand/or cooperativity even at permissive temperatures and losesits operator-binding ability very rapidly above 25°C. Comparativestudies of fluorescence and CD spectra, sulfhydryl group reactivityand elution behaviour in size-exclusion HPLC of both wild-typeand ts2-mutant repressors at permissive and non-permissive temperaturessuggest that the C-terminal domain of the ts2 repressor carryinga K224E substitution has a structure that does not favor tetramerformation at non-permissive temperatures.     

Changes in activity of porcine phospholipase A2 brought about by charge engineering of a major structural element to alter stability

We have modified the stability of porcine phospholipase A₂ bycharge engineering. The mutations are situated at the N-terminalof a major helix and are N89D and N89D/E92Q. This engineeringhas significantly altered the activity of the enzyme to aggregatedand monomeric substrates. A N89D/E92K mutant is more stablebut considerably less active than wild type. An N89D mutantis more stable and of similar activity to wild type. The substantialchange in activity may be due to direct interaction of residue92 with aggregated substrate or may be via second calcium binding.Second calcium binding may be more probable as activity againstmonomers is also affected. Additional calcium binding may thereforebe an important way of manipulating the activity of phospholipaseA₂.     

Second-generation octarellins: two new de novo ({beta}/{alpha})8 polypeptides designed for investigating the influence of {beta}-residue packing on the {alpha}/{beta}-barrel structure stability

The sequence of octarellin I, the first de novo (ß/)₈polypeptide, was revised according to several criteria, amongothers the symmetry of the sequence, ß-residue volumeand hydrophobicity, and charge distribution. These considerationsand the overall conclusions drawn from the first design ledto two new sequences, corresponding to octarellins II and III.Octarellin II retains perfect 8-fold symmetry. Octarellin IIIhas the same sequence as octarellin II, except for the ß-strandswhich exhibit a 4-fold symmetry. The two proteins were producedin Escherichia coli. Infrared and CD spectral analyses of octarellinsII and III reveal a high secondary structure content. Non-denaturinggel electrophoresis, molecular sieve chromatography and analyticalultracentrifugation suggest that both of these second-generationartificial polypeptides exist as a mixture of a monomer anda dimer form. Octarellins II and III are at least 10 times moresoluble than octarellin I. Ureainduced unfolding followed byfluorescence emission suggests that the tryptophan residues,designed to be buried in the (ß/)₈, are indeed packedin the hydrophobic core of both proteins. However, octarellinIII displays a higher stability towards urea denaturation, indicatingthat introducing 4-fold symmetry into the ß-barrelmight be important for stability of the overall folding.     

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.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor {alpha}

A gene encoding human tumour necrosis factor (TNF-) has beenchemically synthesized, cloned and expressed to yield a biologicallyactive protein in Escherichia coli. The 480-bp gene was assembledby enzymic ligation of 32 oligonucleotides, cloned directlyinto M13mp18 for sequence verification and expressed in thebroad host range high-level expression vector pMMB66EHST. Expressedrecombinant TNF- was shown to have the correct molecular weight,processed N-terminal sequence, antibody cross-reactivity andtumour cell killing activity. The expression product of thesynthetic gene has been purified to homogeneity by a two-stepion-exchange procedure and the purified material shown to beactive.     

A holistic approach to protein structure alignment

A method of protein structure comparison developed previouslyis extended to incorporate other aspects of protein structurein addition to the inter-atomic vectors on which it was originallybased. Each additional aspect, which included hydrogen bonding,solvent exposure, torsional angles and sequence, was introducedseparately and evaluated for its ability to improve alignmentquality. The components were then combined, suitably weighted,to produce a more holistic comparison method. The method wastested on a group of remotely related ß/ type proteinsthat share a common feature in their overall chain fold. Theresults indicated that while the original inter-atomic vectorcomponent was sufficient to give the correct alignment of mostpairs of topologically equivalent proteins, the inclusion ofhydrogen bonds, torsion angles and a measure of solvent exposureled to improvements in the more difficult comparisons. Considerationof amino acid properties, including hydrophobicity, had no beneficialeffect. The failure of the latter component was not unexpectedconsidering the almost total lack of sequence similarity amongthe proteins considered.     

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.     

Solution structure and dynamics of a serpin reactive site loop using interleukin 1 as a presentation scaffold

Human interleukin-1ß (IL1ß) was used as a presentationscaffold for the characterization of the reactive site loop(RSL) of the serpin 1-antitrypsin (A1AT), the physiologicalinhibitor of leukocyte elastase. A chimeric protein was generatedby replacement of residues 50–53 of IL1ß, correspondingto an exposed reverse turn in IL1ß, with the 10-residueP5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin,AT-IL) inhibits elastase specifically and also binds the IL1ßreceptor. Multinuclear NMR characterization of AT-IL establishedthat, with the exception of the inserted sequence, the structureof the IL1ß scaffold is preserved in the chimera. Thestructure of the inserted RSL was analyzed relative to thatof the isolated 10-residue RSL peptide, which was shown to beessentially disordered in solution. The chimeric RSL was alsofound to be solvent exposed and conformationally mobile in comparisonwith the IL1ß scaffold, and there was no evidence of persistinginteractions with the scaffold outside of the N- and C-terminallinkages. However, AT-IL exhibits sigificant differences inchemical shift and NOE patterns relative to the isolated RSLthat are consistent with local features of non-random structure.The proximity of these features to the P1-P1' residues suggeststhat they may be responsible for the inhibitory activity ofthe chimera.     

C-terminal truncations of a thermostable Bacillus stearothermophilus {alpha}-amylase

A series of truncated proteins from a thermostable Bacillusstearothermophilus -amylase was prepared to study the importanceof the extension in the C-terminus compared with other liquefyingBacillus -amylases. The mutations introducing new translationtermination sites shortened the 515 amino acid residue-longwild type enzyme by 17, 32, 47, 73 or 93 residues. The longerthe truncation, the lower the specific activity of the enzyme.Only the two longest mutant proteins were active: the specificactivity of the 498 residue variant was 97% and protein 483was 36% that of the parental enzyme. The K_m values of starchhydrolysis changed from 1.09 for wild type enzyme to 0.35 and0.21 for mutants 498 and 483, respectively, indicating alteredsubstrate binding. The mutant enzymes had almost identical pHand temperature optima with the wild type amylase, but enhancedthermal stability and altered end product profile. The consequencesof the truncation to the structure and function of the enzymeswere explored with molecular modeling. The liquefying amylasesseem to require {small tilde}480 residues to be active, whereasthe C-terminal end of B.stearothermophilus amylase is requiredfor increased activity.     

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.     

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.     

Isolated transmembrane helices arranged across a membrane: computational studies

A computational procedure for predicting the arrangement ofan isolated helical fragment across a membrane was developed.The procedure places the transmembrane helical segment intoa model triple-phase system `water–octanol–water';pulls the segment through the membrane, varying its `global'position as a rigid body; optimizes the intrahelical and solvationenergies in each global position by `local' coordinates (dihedralangles of side chains); and selects the lowest energy globalposition for the segment. The procedure was applied to 45 transmembranehelices from the photosynthetic reaction center from Rhodopseudomonasviridis, cytochrome c oxidase from Paracoccus denitrificansand bacteriorhodopsin. In two thirds of the helical fragmentsconsidered, the procedure has predicted the vertical shiftsof the fragments across the membrane with an accuracy of –0.15± 3.12 residues compared with the experimental data.The accuracy for the remaining 15 fragments was 2.17 ±3.07 residues, which is about half of a helix turn. The procedurepredicts the actual membrane boundaries of transmembrane helicalfragments with greater accuracy than existing statistical methods.At the same time, the procedure overestimates the tilt valuesfor the helical fragments.     

Model complexes of tumor necrosis factor-{alpha} with receptors Rl and R2

The biological activities of tumor necrosis factor- (TNF-) aremediated by two different receptors, TNFR1 and TNFR2. To analyzethe receptor binding site(s) of TNF-, molecular models havebeen built of the complexes of TNF- with the extracellular regionsof receptors Rl and R2, based on the known crystal structuresof TNF- and lymphotoxin bound to Rl. The model structure ofR2 from residues 18-160 was built by analogy to the crystalstructure of Rl in complex with lymphotoxin. The amino acidsequences of Rl and R2 show 27.5% identity over this regionand were aligned with five insertions and three deletions. Thereare 18 conserved cysteines that form disulfides. R2 has lostone pair of cysteines compared with Rl, but two new cysteineswere modeled as forming a new disulfide bond. Both symmetricand asymmetric trimers of TNF- were used to model the complexeswith TNFR1 and R2. An analysis of differences in the model complexesshowed good agreement with data on the differential bindingof TNF mutants to its two receptors.     

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study

Proline residues are commonly found in putative transbilayerhelices of many integral membrane proteins which act as transporters,channels and receptors. Intramembranous prolines are often conservedbetween homologous proteins. It has been suggested that suchintrahelical prolines provide liganding sites for cations viaexposure of the backbone carbonyl oxygen atoms of residues i-3and i-4 (relative to the proline). Molecular modelling studieshave been carried out to evaluate this proposal. Bundles ofparallel proline-kinked helices are considered as simplifiedmodels of ion channels. The energetics of K⁺ ion-helix bundleinteractions are explored. It is shown that carbonyl oxygensexposed by the proline-induced kink and at the C-terminus ofthe helices may provide cation-liganding sites. ‘Hybrid’bundles of antiparallel helices, only some of which containproline residues, are considered as models of transport proteins.Again, praline-exposed carbonyi oxygens are shown to be capableof liganding cations. The roles of -helix dipoles and of thegeometry of helix packing are considered in relation to cation-bundleinteractions. Implications with respect to modelling of ionchannel and transport proteins are discussed     

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha

Successful approaches to protein engineering required that thedesired analogs be easily and rapidly obtained in sufficientquantities and purities for unambiguous structural and functionalcharacterizations. Chemical synthesis is the method of choicefor engineering small peptides. We now demonstrate that withimproved methodologies and instrumentation, total chemical synthesiscan be used to produce a small protein in a form suitable forengineering studies. Active human transforming growth factor-alpha(TGF-), a 50 amino acid long protein with three disulfide bonds,has been synthesized and purified in multiple tens of mg amountsin <7 days. The purified human TGF- migrated as a singleband on SDS–polyacrylamide gels, ran as a single sharpmajor band at pI = 6.2 on isoelectric focusing gels, displayedan MW = 5546.2 (Th.5546.3) by mass spectrometry, contained threedisulfide bonds and had EGF receptor binding, mitogenic andsoft agar colony formation activities. The locations of disulfidebonds were found to be analogous to those found in epidermalgrowth factor (EGF) and in human TGF- expressed in bacteria.     

Conformation equilibria of valine studies by dynamics simulation

The conformational probability distribution of a valine residuein the valine dipeptide and of the valine side chain in an -helix,as well as the change in helix stability for replacing alaninewith valine, has been calculated by molecular dynamics simulationsof explicitly hydrated systems: dipeptide, tetrapeptide and10-, 14- and 18-residue oligoalanine helices. All computed free-energydifferences are means from at least eight separate slow-growthsimulations, four in each direction and are reported with theirroot-meansquare deviations. Different values for the changein free energy of folding (G°) have been calculated withthe use of forcefields having an all-atom and a central-atomrepresentation of methyl groups, etc. The value obtained withthe all-atom forcefield agrees well with new experimental values(3 kJ/mol = 0.7 kcal/mol). Furthermore, the most stable valineside-chain rotamer in the helix is different for these two representations.The most stable rotamer for the all atom conformation is thesame one that predominates for valines in a-helices in proteinsof known conformation. The lower conformational freedom of thevaline side chain in the helix contributes 1 kJ/mol to the differencein stability computed with the all-atom potential; unfavorableinteractions of the side chain with the helix, even in the moststable conformation, further increase G°.     

Insertion of an elastase-binding loop into interleukin-1{beta}

The protease-binding sequence EAIPMSIPPE from 1-antitrypsinhas been inserted into the cytokine interleykin-1ß,replacing residues 50–53. The resulting mutant proteinwas cleaved specifically at a singly site by elastase and chymotrypsin,but not by trypson. The cleavage by elastase was shown to bebetween Met and Ser of the inserted loop. In contrast, wild-typeinterleukin is not sus-ceptible to cleavage by any of theseenzymes. The mutant protein acts as an inhibitor of elastase,with a K1 of 30 µM. The wild type displays no such inhibitoryactitvity. The overall structure of the mutant, as demonstratedbyu CD, appears to be indistinguishabel from that fo the wildtype. These results indicate that the protease-binding regionfo 1-antitrypson can be recognized and is active even withinthe context of an entirely differentproteinstructure. Giventhat interleukinm-1ß binds to, and is intenalizedby, many types of cells, this hybrid protein also demonstratesthe feasibility of using interleukin-1ß as a deliverysystem for useful therapeutic agents.