Improved insulin stability through amino acid substitution

Insulin analogs designed to decrease self-association and increaseabsorption rates from subcutaneous tissue were found to havealtered stability. Replacement of H^B10 with aspartic acid increasedstability while substitutions at B²⁸ and/or B²⁹ were eithercomparable to insulin or had decreased stability. The principalchemical degradation product of accelerated storage conditionswas a disulfidelinked multimer that was formed through a disulfideinterchange reaction which resulted from ß-eliminationof the disulfides. The maintenance of the native state of insulinwas shown to be important in protecting the disulfides fromreduction by dithiothreitol and implicitly from the disulfideinter change reaction that occurs during storage. To understandhow these amino acid changes alter chemical stability, the intramolecularconformational equilibria of each analog was assessed by equilibriumdenaturation. The Gibbs free energy of unfolding was comparedwith the chemical stability during storage for over 20 analogs.A significant positive correla tion (R²=0.8 and P < 0.0005)exists between the conformational stability and chemical stabilityof these analogs, indicating that the chemical stability ofinsulin's disulfides is under the thermodynamic control of theconformational equilibria.     

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30

It was previously demonstrated that insulins to which positivecharge has been added by substituting B13 glutamic acid witha glutamine residue, B27 threonine with an arginine or lysineresidue, and by blocking the C-terminal carboxyl group of theB-chain by amidation, featured a prolonged absorption from thesubcutis of rabbits and pigs after injection in solution atacidic pH. The phenomenon is ascribed to a low solubility combinedwith the readiness by which these analogs crystallize as theinjectant is being neutralized in the tissue. However, acidsolutions of insulin are chemically unstable as A21 asparagineboth deamidates to aspartic acid and takes part in formationof covalent dimers via -amino groups of other molecules. Inorder to circumvent the instability, substitutions were introducedin position A21, in addition to those in B13, B27 and B30, challengingthe fact that A21 asparagine has been conserved in this positionthroughout the evolution. Biological potency was retained whenglycine, serine, threonine, aspartic acid, histidine and argininewere introduced in this position, although to a varying degree.In the crystal structure of insulin a hydrogen bond bridgesthe -nitrogen of A21 with the backbone carbonyl of B23 glycine.In order to investigate the importance of this hydrogen bondfor biological activity a gene for the single-chain precursorB-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 prolinewas synthesized. However, this single-chain precursor failedto be properly produced by yeast, pointing to the formationof this hydrogen bond as an essential step in the folding process.The stability of the A21-substituted analogs in acid solutions(pH 3–4) with respect to deamidation and formation ofdimers was {small tilde}5–10 times higher than that ofhuman insulin in neutral solution. The rate of absorption ofmost insulins is decreased by increasing the Zn² concentrationof the preparation. However, one analog with A21 glycine showedfirst-order absorption kinetics in pigs with a half-life of{small tilde}25 h, independent of the Zn² concentration. Theday-to-day variation of the absorption of this analog was significantlylower than that of the conventional insulin suspensions, a propertythat might render such an insulin useful in the attempts toimprove glucose control in diabetics by a more predictable deliveryof basal insulin.     

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry

The production of semisynthetic human insulin for therapeuticpurposes is of considerable importance. During trypsin-catalysedtransformation of pig insulin into an ester of insulin of humansequence, the alanyl residue at position B³⁰ is removed andreplaced with an esterified residue of threonine. We have carriedout this transformation in a medium enriched in ¹⁸OH₂ and studiedthe product by MS. In contrast to a previous report, we findthat incorporation of label into the B²⁹–B³⁰ peptide bondoccurs during the transformation with threonine methyl esterin aqueous N, N-dimethylacetamide. Quantitative data are presentedand the implications of these findings are discussed.     

The semisynthesis of octadeutero-PheB1-octadeutero-ValB2]-procine insulin and its characterization by mass spectrometry

Insulin analogues labelled with stable isotopes (e.g. deuterium,¹⁸O, ¹⁵N, etc.) are authentic (the native structure is rigorouslymaintained), non-radioactive (preferred for injection into man)and can easily be distinguished from endogenous insulin by massspectrometry by virtue of their molecular masses. Appropriatecombinations of amino-protecting groups (methylsulphonylethyloxycarbonyland t-butoxy carbonyl), Edman degradation and chemical couplingwere used to produce [octadeutero-Phe^B1]-porcine insulin and[octadeutero-Phe^B1-octadeutero-Val^B2]-porcine insulin. The analogueswere characterized by electrospray ionization mass spectrometry.Standard mixtures of labelled and unlabelled insulins were successfullystudied by mass spectrometry. Isotope dilution mass spectrometrycould therefore provide a useful direct measure of insulin undertrue physiological conditions, without many of the drawbacksof existing methods. In this regard, the analogue with 16 deuteriumswas more suitable than the octadeuterated analogue, since thegreater mass difference between the labelled and unlabelledforms enabled a lower mass spectrometric resolution to be used,resulting in higher sensitivity     

Stabilization of lysozyme by the introduction of Gly-Pro sequence

Three mutant lysozymes where the Asp¹⁰¹ – Gly¹⁰² sequenceof lysozyme was converted to Asp¹⁰¹–Pro¹⁰², Gly¹⁰¹–Pro102and Pro¹⁰¹–Gly¹⁰² were prepared to investigate the effectof proline residues on the stabilization of proteins. The freeenergy changes of lysozymes for the unfolding in aqueous solutionat pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/molfor wild type, Asp¹⁰¹Pro¹⁰², Gly¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozymerespectively. When the energy level in the unfolded state ofwild type lysozyme was fixed at a standard level, the energylevels in the folded state of Asp¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰²lysozymes were found to be higher than that of wild type lysozymeon the basis of G_D(H₂O) and entropy losses of their polypeptidechains in the unfolded state. The presence of some strain inthe folded state of these lysozymes was supported by both thecalculation of conformational energy for a trans-L-prolyl residue[Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105–120] and the analysis of structures of energy-minimizedmutant lysozymes. Therefore, it is concluded that the formationof the Gly-Pro sequence is effective in avoiding possible strainin the folded state of a protein caused by the introductionof proline residue(s).     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

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.     

Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain

Hydrophilic insulins, more positively charged than human insulinat neutral pH, have been prepared by substitution with basicamino acids at the termini of the B-chain and by blocking theC-terminal carboxyl group of the B-chain. The iso-electric pHof the insulin is thereby moved from 5.4 towards physiologicallevels. Slightly acid solutions of derivatives, in which chargehas been added in the C-terminus of the B-chain, have a prolongedaction in vivo, in particular if the carboxyl group is blocked.It is found that the prolong ed-acting hydrophilic insulinscrystallize instantly when the pH is adjusted to 7. The prolongedaction is ascribed to this readiness to crystallization combinedwith a low solubility, which may be further decreased by Increasedconcentration of zinc ions. Hydrophobic insulins have a prolongedaction independent of the site of substitution even if the derivativeis soluble at physiological pH. Some derivatives were preparedfrom porcine Insulin by tryptic transpeptidatlon. N-terminalB-chain substituted insulins were prepared by alkylation ofa biosynthetic single-chain insulin precursor, followed by tryptictranspeptidation rendering the double chain insulin derivative.The observed blood glucose lowering in the rabbits implies thatneither N- nor C-terminal B-chain substitution results in substantialdeterioration of biological potency. An index for the degreeof protraction based on the blood glucose data is used to comparethe insulins.     

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.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positivecharge has been added by substitutions in position B30, thusraising the isolectric point towards pH 7, had a prolonged actionwhen injected as slightly acidic solutions because such derivativescrystallize very readily upon neutralization. Positive chargehas now been added by substituting the B13 and A17 glutainicacid residues with glutamines and B27 threonine with lysineor arginine. These substitutions were introduced by site-specificmutagenesis in a gene coding for a single-chain insulin precursor.By tryptic transpeptidation the single-chain precursors weretransformed to the double-chain insulin structure, concomitantlywith incorporation of residue B30. Thus insulins combining B13glutamine, A17 glutamine and B27 lysine or arginine with B30threonine, threonine amide or lysine amide were synthesized.The time course of blood glucose lowering effect and the absorptionwere studied after subcutaneous injection in rabbits and pigs.The prolonged action of B30-substituted insulins s marked lyenhanced by B27 lysine or arginine substitutions and by B13glutamine. The B27 residue is located on the surface of thehexamer, so a basic residue in this position presumably promotesthe packing of hexainers at neutral pH. The B13 residues clusterin the centre of the hexainer. When the electrostatic repulsiveforces from six glutamic acid residues are abolished by substitutionwith glutamine, a stabilization of the hexamer can he envisaged.The biological potency of insulins was measured in the freefat cell assay and in the mouse blood glucose assay test. Apotency factor could be fitted to each substitution, so thatthe potency of analogs with two or more substitutions can beestimated by multiplication of the corresponding potency factors.A charge-indifferent substitution, B8 glycine to serine, resultedin insulins that crystallize well but have low potencies. Alate elution in gradient r.p.-h.p.l.c. indicates that hydrophobicamino acid residues have been exposed as a result of this B8substitution. This most likely results from distortion of the-helix cominenc ing at residue B7, permitted only by B8 glycinewith dihedral angles (, ) of a D-amino acid.     

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Comparison of scales of amino acid side chain properties by conservation during evolution of four proteins

As the amino acid sequence of a given protein changes alongthe phylogenetic tree, enough of the overall folding patternmust be conserved to ensure that the protein still fulfils itsbiological function. Eighteen published scales which tabulatevarious side chain properties are compared here by computingthe variance of each scale when applied to each of several proteinfamilies. The conservation of each scale of side chain propertiesis examined for the 20 627 residues in 60 mammalian myoglobins,31 mammalian ribonucleases, insulin A and B chains (29 sequenceseach), 29 vertebrate and 28 plant cytochrome c's. Those scaleswhich are the most highly conserved through the evolution ofeach protein family may well be the best predictors of proteinfolding patterns. The mean-area-buried scale and the optimizedmatching hydrophobicities scale are more conserved than otherscales. An additional result is the relatively poor conservationacross evolution of the Chou-Fasman secondary structure predictors.     

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogenactivator (t-PA) is in part associated with the presence ofthe kringle 2 domain in t-PA. Within this kringle 2 domain alysyl-binding site has been described. The plasminogen to plasminconversion by urokinase-type plasminogen activator (u-PA), incontrast to that of t-PA, is not enhanced in the presence offibrin. Within the u-PA kringle domain no lysyl-binding siteis found. To study whether introduction of a lysyl-binding sitein the u-PA kringle domain will make u-PA a fibrin-dependentplasminogen activator, three stretches of amino acid residuesof the u-PA kringle domain (A²⁸-Q³³, D⁵⁵-N⁵⁷ and G⁶⁷-V⁷²) weresubstituted by three stretches of amino acids from the correspondingpositions of the kringle 2 domain of t-PA (M²⁸-K³³, D⁵⁵-D⁵⁷and N⁶⁷-W⁷²). These changes resulted in the creation of thelysyl-binding site consensus of the kringle 2 domain (K³³, D⁵⁵,D⁵⁷, W⁶² and W⁷²) in the u-PA kringle. However, the resultingu-PA mutant did not interact with lysyl-Sepharose, nor did itdisplay fibrin-enhanced plasminogen activation in the presenceof soluble fibrin mimic. When the kringle domain of u-PA wasreplaced by the kringle 2 domain of t-PA, similar results wereobtained. The hybrid protein hardly interacted with lysyl-Sepharoseand the plasminogen activation was not enhanced in the presenceof fibrin mimic However, the N-terminal fragment isolated fromthis hybrid molecule (consisting of growth factor domain andkringle 2 domain) did interact with lysyl-Sepharose, suggestingthat in the hybrid molecule a functional lysyl-binding siteis present but not operational. Indeed, lysine analogue (e-amino-caproicacid) sensitive binding of isolated t-PA kringle 2 domain tou-PA could be observed. The modified u-PA kringle, the wildtype u-PA kringle and the kringle 2 of the u-PA hybrid werealso placed N-terminal of the protease domain of t-PA. As expected,the t-PA mutant consisting of the kringle 2 domain and the proteasedomain bound to lysyl-Sepharose and showed fibrin-dependentplasminogen activation. Further, the hybrid molecule consistingof the u-PA kringle placed N-terminal of the t-PA protease domaindid not display these features. Introduction of the modifiedu-PA kringle N-terminal of the t-PA protease domain resultedin a very weak interaction with lysyl-Sepharose. Despite thehigh overall similarity in primary structure of the modifiedu-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogenactivation of this hybrid molecule was observed. The above-mentionedresults question the concept that the structural auto-nomousdomains within hybrid plasminogen activators t-PA and u-PA functionas autonomous domains and suggest that interactions betweenthe kringle and the protease domain in hybrid molecules stronglyinfluences their functional features     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAF^–B (coding for all amino acids besides phenylalanine)was obtained by ‘cassette mutagenesis’ from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAF^–B in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AF^–B and AHB genes in pBIO52.Complete translation of the expressed genes AB, AF^–B andAHB in either the in vitro or in vivo systems could be shownby using ³⁵S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increasedor decreased to alter the electrostatic interaction betweenenzyme and substrate in the lytic action of human lysozyme usingsite-directed mutagenesis. The amino acid substitutions accompanyingeither the addition or the removal of two units of positivecharge have shifted the optimal ionic strength (NaCl concentrationin 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticuscell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively.In addition to the change in ionic strength–activity profile,the pH–activity profile and the effect of a polycationicelectrolyte, poly-L-Lys-HCl, on the lytic activity were significantlychanged. Owing to the shifts in both ionic strength profilesand pH profiles the Arg⁷⁴/Arg¹²⁶ mutant has become a bettercatalyst than wild-type enzyme under the conditions of highionic strength and high pH, and the Gln⁴¹/Ser¹⁰¹ mutant hasbecome a better catalyst under the conditions of low ionic strengthand low pH.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B()

P26olf from olfactory tissue of frog, which may be involvedin olfactory transduction or adaptation, is a Ca²⁺-binding proteinwith 217 amino acids. The p26olf molecule contains two homologousparts consisting of the N-terminal half with amino acids 1–109and the C-terminal half with amino acids 110–217. Eachhalf resembles S100 protein with about 100 amino acids and containstwo helix–loop–helix Ca²⁺-binding structural motifsknown as EF-hands: a normal EF-hand at the C-terminus and apseudo EF-hand at the N-terminus. Multiple alignment of thetwo S100-like domains of p26olf with 18 S100 proteins indicatedthat the C-terminal putative EF-hand of each domain containsa four-residue insertion when compared with the typical EF-handmotifs in the S100 protein, while the N-terminal EF-hand ishomologous to its pseudo EF-hand. We constructed a three-dimensionalmodel of the p26olf molecule based on results of the multiplealignment and NMR structures of dimeric S100B(ßß)in the Ca²⁺-free state. The predicted structure of the p26olfsingle polypeptide chain satisfactorily adopts a folding patternremarkably similar to dimeric S100B(ßß). Each domainof p26olf consists of a unicornate-type four-helix bundle andthey interact with each other in an antiparallel manner formingan X-type four-helix bundle between the two domains. The twoS100-like domains of p26olf are linked by a loop with no sterichindrance, suggesting that this loop might play an importantrole in the function of p26olf. The circular dichroism spectraldata support the predicted structure of p26olf and indicatethat Ca²⁺-dependent conformational changes occur. Since theC-terminal putative EF-hand of each domain fully keeps the helix–loop–helixmotif having a longer Ca²⁺-binding loop, regardless of the four-residueinsertion, we propose that it is a new, novel EF-hand, althoughit is unclear whether this EF-hand binds Ca²⁺. P26olf is a newmember of the S100 protein family.