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.     

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.     

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.     

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     

Improvement of turn structure prediction by molecular dynamics: a case study of {alpha}1-purothionin

Because of the problems in predicting a correct conformationfor loop regions in homology-based prediction, disagreementsare often found between the predicted models and the refinedX-ray structures of the same protein in loop regions. Such asituation has been encountered for ₁-purothionin (₁-PT). Hence,attempts have been made to improve the predicted model of ₁PTby limited molecular dynamics using both AMBER and XPLOR. Withmolecular dynamics, the previously predicted incorrect turnregion reverts to the correct conformation as seen in the X-rayrefined structure. In contrast to the model which is not subjectedto molecular dynamics, the improved model refines with the X-raydata of ₁PT in fewer cycles, without any manual rebuilding andwith comparable or better refinement statistics. Also, the improvedmodel serves as a better starting model in the determinationof the structure with the molecular replacement methods.     

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.     

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.     

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.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster _1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507A, N507D and N507S) have alsobeen simulated; among these, the N507S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the _1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.     

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.     

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.     

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     

Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or Asp119

In the cross-linking reaction of lysozyme between Leu¹²⁹ (-COO^–)and Lys¹³ (-NH₃⁺ using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC), a side reaction of the peptide bond inversionfrom to ß between A and Gly¹⁰² was greatly reducedby addition of ß-(1,4)-linked trimer of N-acetyl-D-glucosamine[(NAG)₃] When methylamine or 2-hydroxyethylamine was furtheradded, the extent of the cross-link formation was decreasedand the derivative where the -carboxyl group of Leu¹²⁹ was modifiedwith the amine was newly obtained. On the other hand, when ammoniawas added, the ß-carboxyl group of Asp¹¹⁹ insteadof the -carboxyl group was mainly amidated. From these results,the presence of a salt bridge between Asp¹¹⁹ and Arg¹²⁵ besidesthat between Lys¹³ and Leu¹²⁹ is proposed. Enzymatic activitiesof the derivatives prepared here indicated that the modificationof the -carboxyl group reduced the activity to {small tilde}90% of that of native lysozyme. Des-Leu¹²⁹ lysozyme, which lacksLeu¹²⁹ also showed {small tilde} 90% of the activity of nativelysozyme. Therefore, the salt bridge between Lys¹³ and Leu¹²⁹may play some role in maintaining the active conformation oflysozyine.     

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.     

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.     

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.     

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.     

Activation of the low oxygen affinity-inducing potential of the Asn108({beta}){->}Lys mutation of Hb-Presbyterian on intramolecular {{alpha}}{{alpha}}-fumaryl cross-bridging

The Asn108ßLys mutation in hemoglobin (HbPresbyterianmutation) endows a low O₂ affinity-inducing propensity to theprotein. Introduction of a fumaryl cross-bridge between itstwo 99 lysine residues also induces a low O₂ affinity into HbA.We have now engineered an -fumaryl cross-bridge into Hb-Presbyterianto determine the synergy or additivity, if any, that can beachieved between these two low O₂ affinity-inducing structuralperturbations. Despite the presence of the additional -aminogroup of Lys108(ß) within the central cavity, the-amino group of Lys99() of deoxy Hb-Presbyterian retained highselectivity for -fumaryl cross-bridging, with an overall efficiencycomparable to that with HbA. The -fumaryl cross-linking of Hb-Presbyterianreduced its O₂ affinity much more significantly than that observedwith HbA, indicating a synergy between the two low O₂ affinity-inducingstructural perturbations. Apparently, the -fumaryl cross-bridgein Hb-Presbyterian activates part of the latent low O₂ affinity-inducingpotential of Lys108(ß) that is generally activatedin the presence of chloride. The synergy between the Asn108(ß)Lysmutation and the -fumaryl cross-bridging was conserved in thepresence of chloride, but not in the presence of DPG. Furthermore,in the presence of chloride and DPG, -fumaryl Hb-Presbyterianaccessed a low O₂ affinity T-state that is accessed by HbA,-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectricfocusing analysis suggested that the -fumaryl cross-linkingof Hb-Presbyterian induces changes in the ionization behaviorof one or more of the functional groups neighboring Lys99()and Lys108(ß) [presumably His103() and/or Glu101(ß)]to compensate for the extra positive charge of Lys108(ß).Molecular modeling studies identified two potential chloridebinding sites per ß dimer within the middle of thecentral cavity of -fumaryl HbA involving residues His103(),Arg104(ß) and Asn108(ß). The affinity ofthese sites is increased in -fumaryl Hb-Presbyterian as a resultof the Asn108(ß)Lys mutation. Thus, the results ofthe present study suggest that the enhanced neutralization ofthe positive charges in the middle of the central cavity ofHb achieved by these two electrostatic modifications, one (the-fumaryl cross-bridge) acting directly and the other (the Presbyterianmutation) acting indirectly through the mediation of chlorideion binding, facilitates the - fumaryl-Hb Presbyterian to accessa low O₂ affinity T-state structure much more readily than eitherHb-Presbyterian or -fumaryl HbA.     

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.