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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-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.     

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.     

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.     

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.     

Production and characterization of anti-human interferon {gamma} receptor antibody fragments that inhibit cytokine binding to the receptor

Three single-chain antibody fragments that recognize the extracellularhuman interferon receptor -chain (IFNR), and inhibit the bindingof human IFN, have been produced in Escherichia coli. Thesefragments are derived from murine anti-receptor monoclonal antibodies,and comprise the variable heavy (V_H) domain linked to the variablelight (V_L) chain through a 15 amino acid linker [(GGGGS)₃].Using surface plasmon resonance technology (BIAcore), the solubleproteins were shown to retain a high affinity for recombinantIFNR, and by radioimmunoassay to possess high inhibitory activitytowards IFN-binding to human Raji cells. The antibody fragmentsmost likely recognize epitopes that overlap the cytokine bindingsite on the receptor surface. Attempts to dissect further theantibodies to isolated V_H- and V_L-chains and to synthetic linearand cyclic peptides derived from the individual complementaritydetermining regions failed to afford fragments with significantIFNR binding affinity. Nevertheless, these native-like variableregion fragments and petidomimetics derived from them are ofinterest in the design of novel IFNR antagonists.     

Structure-function analysis of human IL-1{alpha}: identification of residues required for binding to the human type I IL-1 receptor

Using oligonucleotide-directed mutagenesis, the binding siteon human interleukin-1 (IL-1) for the human type I IL-1 receptor(IL-1R) has been analyzed. Substitution of seven amino acids(Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resultedin a significant loss of binding to the receptor. Based on crystallographicinformation, the side chains of these residues are clusteredin one region of IL-1 and exposed on the surface of the protein.Five of the residues in the IL-1 binding site align with thebinding residues previously determined in human IL-1ß,demonstrating that the type I IL-1R recognizes homologous regionsin both ligands. Unexpectedly, only three of the aligned residuesare identical between IL-1 and IL-1ß. These observationssuggest that the composition of contact residues in the bindingsite is unique for each ligand–receptor complex in theIL-1 system.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.     

Composition analysis of {alpha}-helices in thermophilic organisms

We present a statistical comparison of the amino acid compositionin a secondary structure element, the -helix, of proteins stableat high temperatures with those which are less so. This studyhas shown that the temperature-dependent Zimm-Bragg helix propagationvalue s is not a good predictor for the helix-forming tendencyof an amino acid in thermostable proteins. However, we haveshown that s, the change in s from 20 to 60°C, accuratelypredicts the direction of the probability shift for 15 aminoacids in thermostable protein a-helices, although it does notpredict the magnitude of that change. The residues tyrosine,glycine and glutamine show a significant increase in residencyin a-helices for thermostable proteins over their nonthermostablecounterparts. Significant decreases in -helix residency occurfor the residues valine, glutamic acid, histidine, cysteineand aspartic acid in proteins from thermophilic organisms. Aromaticinteractions, hydrogen bonding and a reduction of charge mayexplain the increase observed for tyrosine and glutamine andthe decrease in glutamic acid and aspartic acid, although packingconsiderations cannot be ruled out The only physical explanationfor the increase in glycine would seem to be its positive svalue     

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.     

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.