The preparation of porcine (Tyr-A14-2H) insulin and its characterization by mass spectrometry

The title compound was prepared by catalytic deiodination of(Tyr-A¹⁴-3-I) insulin with deuterium gas. Under special conditions(large excess of PdO in CH₃OD/D₂O at pH 9.2) developed to minimizeproblems due to poisoning of the catalyst, deuterated insulinwas produced in yields of about 35% after purification by reversedphase HPLC. For analysis, the deuterated insulin was digestedwith V8 protease and was shown by mass spectrometry to haveincorporated deuterium to an extent of 96.6 atom %, exclusivelyin a pentapeptide containing Tyr A¹⁴. The title compound shouldprove useful to those workers studying insulin by mass spectrometry,and use of the method with tritium gas in place of deuteriumshould permit the preparation of a specifically labelled radioactiveinsulin analogue which behaves identically to the natural hormone.     

Altering the association properties of insulin by amino acid replacement

The importance of Pro^B28 and Lys^B29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B²⁸ and B²⁹ Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B²⁸is critical for high-affinity self-association. Removal of Pro^B28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro²⁸, greatly reduced association. The largestdisruption to association was achieved by replacing Lys^B29 withPro and varying the amino acid at B²⁸ Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids.     

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.     

Expression in insect cells and purification of a catalytically active recombinant human gastric lipase

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (Baculogold^TM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High Five^TM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High Five^TM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDS–PAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (Intralipid^TM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively.     

Engineering a uniquely reactive thiol into a cysteine-rich peptide

Cysteine mutagenesis for the purpose of chemical labelling wasapplied to the K⁺ channel neurotoxin charybdotoxin, a 37-residuepeptide with six functionally essential cysteines. An additional‘spinster cysteine’ was introduced at a positionfar away in space from the toxin's known interaction surfacewhere it contacts its K⁺ channel receptor. Despite the presenceof the extra unpaired cysteine residue,the toxin still foldsefficiently and may be labelled by fluorescent and radioactivereagents to give a functionally competent toxin.     

Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022

BM 06.022 is a t-PA deletion variant which comprises the kringle2 and the protease domain. Production of BM 06.022 in Escherichiacoli leads to the formation of inactive inclusion bodies, whichhave to be refolded by an in vitro refolding process to achieveactivity and proper structure of the domains. We analysed thebiochemical properties of BM 06.022 to obtain some informationabout the structure of kringle 2 and the protease as comparedwith the structure of these domains in the intact t-PA molecule.The kinetic analysis of the amidolytic activity of BM 06.022and CHO-t-PA yielded similar values for k_cat (13.9 s^-1and 11.4s^-1for the single chain forms and 33.9 s^-1and 27.1 s^-1for thetwo chain forms of BM 06.022 and CHO-t-PA, respectively) andfor k_m, (2.5 mM and 2.1 mM for the single chain forms and 0.5mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA,respectively). BM 06.022 and CHO-t-PA have the same plasminogenolyticactivity in the absence of CNBr fragments of fibrinogen. However,BM 06.022 has a lower plasminogenolytic activity in the presenceof CNBr fragments of fibrinogen and a lower affinity to fibrinas compared with CHO-t-PA. The affinity of BM 06.022 for fibrinis completely suppressed by 0.3 mM eaminocaproic acid, whilethe intact t-PA has a residual affinity of 30%. The dissociationconstants for the interaction with the lysine analogue e-aminocaprokacid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA,respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibitedby PAI-1 in a similar manner     

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     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

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.     

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.     

The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer

The subunit structure of human macrophage migration inhibitoryfactor (MIF) has been studied by preliminary X-ray analysisof wild-type and selenomethionine-MIF and dynamic light scattering.Crystal form I of MIF belongs to space group P2₁2₁2₁ and isgrown from 2 M ammonium sulfate at pH 8.5. A native data sethas been collected to 2.4 Å resolution. Self-rotationstudies and V_m values indicate that three molecules per asymmetricunit are present A data set to 2.8 Å resolution has beencollected for crystal form II, which belongs to space groupP3¹21 or P3²21 and grows from 2 M ammonium sulfate, 2% polyethyleneglycol (average molecular mass 400), 0.1 M HEPES, pH 7.5. Three,four, five or six monomers in the asymmetric unit are consistentwith V_m values for this crystal form. Analysis of crystal formII containing selenomethionine-MIF indicates nine selenium sitesare present per asymmetric unit. Dynamic light scattering ofMIF suggests that the major form of the protein in solutionis a trimer. The results of these studies are in contrast toprevious reports indicating that MIF is a monomer or dimer.The subunit arrangement of MIF is similar to that of tumor necrosisfactor and suggests that signal transduction might require trimerizationof receptor subunits.     

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.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

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.     

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.     

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants

We have constructed an expression vector that leads to secretionof the whole Fc of human immunoglobulin E (hIgE-Fc) from mammaliancells at levels up to 100 mg/l of culture. Two surface glycosylationsites at Asn265 and Asn371 have been changed to glutamine, toobtain a more homogeneous preparation of hIgE-Fc for structuralstudies. Comparison of wild-type and mutant products revealedthat Asn371 is rarely glycosylated in Chinese hamster ovarycells. Both the double mutant and wild-type hIgEFc bind to thehigh-affinity IgE receptor, FcRI, with about the same affinityas myeloma IgE (K_a in the range 10¹⁰–10¹¹ M^–1),and were able to sensitize isolated human basophils for anti-IgEtriggering of histamine release. However, only the double mutanthIgE-Fc approached the affinity of myeloma IgE for the low-affinityreceptor, FcRII (K_a = 7.3x10⁷ M^–1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (K_a = 4.1x10⁶M^–1).     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDS–PAGE ‘snapshot’ analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZ–GST::rFosLZ–GSTheterodimers whereas rJunLZ–GST::rFosLZ and rJunLZ::rFosLZ–GSTformed readily. Furthermore, rJunLZ–GST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (K_a >10⁶ M^-1) with no higher multimeric forms. rFosLZ–GST weaklyassociates beyond a dimer (K_a {small tilde}4x10⁵ M^-1) and rJunLZ–GSTassociates indefinitely (K_a {small tilde}4x10⁶ M^-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired.     

Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions

Assumptions about the dependence of protein unfolding on theconcentration of urea have been examined by an extensive surveyof the equilibrium unfolding of barnase and many of its mutantsmeasured by urea denaturation and differential scanning calorimetry.The free energy of equilibrium unfolding and the activationenergy for the kinetics of unfolding of proteins are generallyassumed to change linearly with [urea]. A slight downward curvatureis detected, however, in plots of highly precise measurementsof logjtu versus [urea] (where k_u is the observed rate constantfor the unfolding of barnase). The data fit the equation logkk_u= logk_u^H₂O* + mk_u^*.[urea] – 0.014[urea]², where mk_u^*is a variable which depends on the mutation. The constant 0.014 was measured directly on four destabilized mutants and wildtype, and was also determined from a global analysis of data from>60 mutants of barnase. Any equivalent deviations from linearityin the equilibrium unfolding are small and in the same region,as determined from measurements on 166 mutants. The free energyof unfolding of barnase, G_U–F, appears significantly largerby 1.6 kcal mol^–1 when measured by calorimetry than whendetermined by urea denaturation. However, the changes in G_U–Fon mutation, G_U–F, determined by calorimetry and by ureadenaturation are identical. We show analytically how, hi general,the curvature in plots of activation or equilibrium energiesagainst [denaturant] should not affect the changes of thesevalues on mutation provided measurements are made over the sameconcentration ranges of denaturant and the curvature is independentof mutation.