Recombinant-derived interleukin-1{alpha} stabilized against specific deamidation

Recombinant-derived human interleukln-1 (IL-1), purified fromEscherichia coli, was resolved by isoelectric focusing on polyacrylamidegels into two species of isoelectric points (pI) 5.45 and 5.20,which constituted 75% and 25% of the total IL-1 protein respectively.The pI 5.45 and pI 5.20 species were separated by chromatofocusingand subjected to N-terminal sequence analysis. The pI 5.45 speciescontained the expected Asn residue at position 36 of the matureprotein sequence whereas the pI 5.20 species contained an Aspresidue at the same position. A mutant protein in which Asn-36was substituted for a Ser residue was isolated from E.coli andshown to be homogeneous on isoelectric focusing analysis witha pI = 5.45. ¹H-n.m.r. and circular dichroism analyses of wild-typeand the mutant IL-1 indicated a similar conformation which wasalso indicated by the identical receptor binding affinitiesof IL-1 with Asn, Asp or Ser in position 36. The mutant proteinwas stabilized against specific base catalysed and temperature-induceddeamidation, and may be more suitable than the wild-type positionfor physical and structural studies.     

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.     

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.     

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.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Altered specificities of genetically engineered {alpha}1 antitrypsin variants

Seven active site variants of human ₁-antitrypsin (₁AT) wereproduced in Escherichia coli following site-specific mutagenesisof the ₁AT complementary DNA. ₁AT (Ala ³⁵⁸), ₁AT (Ile³⁵⁸ and₁AT (Val³⁵⁸), were efficient inhibitors of both neutrophil andpancreatic elastases, but not of cathepsin G. ₁AT (Ala³⁵⁸, Val³⁵⁸)and ₁AT (Phe³⁵⁸ specifically inhibited pancreatic elastase andcathepsin G respectively. The most potent inhibitor of neutrophilelastase was ₁AT (Leu³⁵⁸), which also proved to be effectiveagainst cathepsin G. The ₁AT (Arg³⁵⁸) variant inactivated thrombinwith kinetics similar to antithrombin III in the presence ofheparin. Electrophoretic analysis showed that SDS-stable highmol. wt complexes were formed between the mutant inhibitorsand the cognate proteases in each case. These data indicatethat effective inhibition occurs when the ₁AT P1 residue (position358) corresponds to the primary specificity of the target protease.Moreover, alteration of the P3 residue (position 356) can furthermodify the reactivity of the inhibitor. Two of the variantshave therapeutic potential: ₁AT (Leu³⁵⁸ may be more useful thanplasma ₁AT in the treatment of destructive lung disorders and₁ (Arg³⁵⁸ could be effective in the control of thrombosis.     

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.     

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.     

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.     

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.     

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.     

Functionally essential, invariant glutamate near the C-terminus of strand {beta}5 in various ({alpha}/{beta})8-barrel enzymes as a possible indicator of their evolutionary relatedness

Twelve different (/ß)₈-barrel enzymes belonging tothree structurally distinct families were found to contain,near the C-terminus of their strand ß5, a conservedinvariant glutamic acid residue that plays an important functionalrole in each of these enzymes. The search was based on the ideathat a conserved sequence region of an (/ß)₈-barrelenzyme should be more or less conserved also in the equivalentpart of the structure of the other enzymes with this foldingmotif owing to their mutual evolutionary relatedness. For thispurpose, the sequence region around the well conserved fifthß-strand of a-amylase containing catalytic glutamate(Glu230, Aspergillus oryzae -amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (/ß)₈-barrels are discussed from both thesequence-structural and the evolutionary point of view, theinvariant glutamate residue being proposed to be a joining featureof the studied group of enzymes remaining from their ancestral(/ß)₈-barrel     

{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.     

The use of alanine scanning mutagenesis to determine the role of the N-terminus of the regulatory chain in the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase

The location of the first seven residues of the regulatory chainof Escherichia coli aspartate transcarbamoylase has been identifiedby X-ray crystallography to be near the binding site of theregulatory nucleotides. In order to determine the function ofthe N-terminus of the regulatory chain of aspartate transcarbamoylasein heterotropic regulation, alanine scanning mutagenesis wasused. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7rwere each replaced with alanine. Analyses of these mutant enzymesindicate that none of these substitutions significantly alterthe catalytic properties of the enzyme. However, three of themutant enzymes, Asp4r Ala, Lys6r Ala and Leu7r Ala, exhibitednotable changes in their response to the regulatory nucleotides,while mutations at Thr2r, His3r and Asn5r exhibited only minorchanges in their heterotropic responses. For the Asp4r Alaenzyme, the responses to ATP and CTP were reduced 30 and 40%respectively, compared with the wild-type enzyme. For the Lys6r Ala enzyme, the response to ATP was reduced 70%, while theCTP response was reduced 50%. In the case of the Leu7r Alaenzyme, a 30 and 20% reduction in response to ATP and CTP respectively,was observed. The synergistk inhibition by UTP in the presenceof CTP for the Lys6r Ala enzyme was reduced 40% compared withthat of the wild type enzyme. For the Leu7r Ala enzyme, thesynergistic inhibition was abolished. In addition, UTP decreasedthe CTP binding affinity of the Leu7r Ala enzyme. Analysisof the kinetic data from these mutant enzymes suggests thatresidues Thr2r, His3r and Asn5r have little effect on the heterotropicmechanism, while residues Asp4r, Lys6r and Leu7r play a moresignificant role in the heterotropic response of the enzymetoward the nucleotides. Furthermore, residue Leu7r appears tobe directly involved in the mechanism for synergistic inhibitionof aspartate transcarbamoylase. In this study alanine scanningmutagenesis has provided a rapid method of identifying thoseresidues in the N-terminal region of the regulatory chain ofaspartate transcarbamoylase important for heterotropic regulation.     

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.     

Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression

Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2genes, display 88% amino acid sequence similarities. The dissimilaritiesof sequence between these two enzymes are primarily localizedwithin four discrete regions of the polypeptides that are separatedby regions of absolute sequence identity. IIA1 specificallyhydroxylates the prototype substrate testosterone at the 7 and6 position with a predominance of 7 metabolite. IIA2, on theother hand, hydroxylates this steroid at eight positions onthe molecule, with one of the most abundant metabolites being15hydroxytestosterone. To determine those amino acids responsiblefor the difference in testosterone hydroxylation specificities,chimeras were constructed between IIA1 and IIA2 cDNAs and expressedin cell culture using vaccinia-virus-mediated cDNA expression.Chimeras, in which the first 355 amino acids correspond to asingle enzyme, maintain the specificity associated with thatenzyme. Of six chimeras which have substitutions between aminoacids 161 and 276, two are inactive and the remaining four givesimilar metabolite profiles, in which both 7 and 15 hydroxylationspecificities have been lost. Two of these four chimeras arediametric apposites, suggesting that modification of eitherthe N-terminal or central regions of the enzymes results inconformational changes that prevent the specific binding interactionsresponsible for the narrow regioselectivity associated withIIA1 and 15-hydroxytestosterone formation associated with IIA2.     

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.     

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.     

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.     

Building protein backbones from C{alpha} co-ordinates

An automatic procedure for building polyalanine backbones fromguiding -carbon positions is presented. Polyalanine backbonesare built based on the geometric restraints of angle N-C-C andthe knowledge of main-chain dihedral angle distributions. Abuilding module constructs a list of polyalanine backbones thatfollow exactly the C trace. Then a selection module selectsone backbone with the largest portion of phi-psi pairs in favouredregions. Several test cases on C coordinates from X-ray refinedstructures give acceptable results. Less than 10% of the peptideplanes are incorrectly built, and the result is not sensitiveto random shift up to 0.5 Å of C coordinates.