Estimating the twist of {beta}-strands embedded within a regular parallel {beta}-barrel structure

The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, T_s, is introduced. From T_s, theß-strand twist (Tw_ß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Tw_ß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy.     

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.     

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.     

Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6

We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL–6 portion of the diphtheria toxin-related interleukin–6(IL–6) fusion toxin DAB₃₈₉-IL–6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL–6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the C–terminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL–6 portion of DAB₃₈₉-IL–6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys⁴⁴⁰, Cys⁴⁴⁶, Cys⁴⁶⁹ or Cys⁴⁷⁹ to Ser respectively, demonstratesthat only the disulfide bridge between Cys⁴⁶⁹ and Cys⁴⁷⁹ isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435–451,which includes Cys⁴⁴⁰ and Cys⁴⁴⁶, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys⁴⁴⁰ and Cys⁴⁴⁶ is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB₃₈₉-IL6(435–451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency.     

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).     

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.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to X-ray crystallographic and NMR data

The native solution structure and dynamics of chymotrypsin inhibitor2 (CI2) have been studied using a long (5.3 ns) molecular dynamics(MD) simulation without any imposed restraints. The majorityof the experimentally observed spin–spin coupling constants,short– and long–range nuclear Overhauser effect(NOE) cross peaks and the amide hydrogen exchange behavior werereproduced by the MD simulation. This good correspondence suggeststhat the major structural features of the protein during thesimulation are representative of the true protein structurein solution. Two water molecules formed hydrogen bond bridgesbetween ß₂ and ß₃, in agreement with X–raycrystallographic data and a recent reassessment of the solutionstructure using time averaged NMR restraints during MD refinement.The active–site loop of the protein displayed the greateststructural changes and the highest mobility. When this loopregion was excluded, the average C r.m.s. deviation of the simulatedsolution structures from the crystal structure was 1.5 from0.5 to 5 3 ns. There is structural heterogeneity in particularregions of the NMR–derived solution structures, whichcould be a result of imprecision or true internal motion. Astudy of the distribution of mobility through the protein allowsus to distinguish between these two alternatives. In particular,deviations in the active–site loop appear to be a resultof heightened mobility, which is also supported by good correspondencebetween calculated and experimental S² N–H order parameters.On the other hand, other ill–defined regions of the NMR-derivedstructures are well defined in the simulation and are probablythe result of a lack of structural restraints (i.e. NOEs), asopposed to reflecting the true mobility.     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Influenza virus M2 protein: a molecular modelling study of the ion channel

The influenza A M₂ protein forms cation-selective ion channelswhich are blocked by the anti-influenza drug amantadine. A molecularmodel of the M₂ channel is presented in which a bundle of fourparallel M₂ transbilayer helices surrounds a central ion-permeablepore. Analysis of helix amphipathicity was used to aid determinationof the orientation of the helices about their long axes. Thehelices are tilted such that the N-terminal mouth of the poreis wider than the C-terminal mouth. The channel is lined byresidues V27, S31 and I42. Residues D24 and D44 are locatedat opposite mouths of the pore, which is narrowest in the vicinityof I42. Energy profiles for interaction of the channel withNa⁺, amantadine-H⁺ and cyclopentylamine-H⁺ are evaluated. Theinteraction profile for Na⁺ exhibits three minima, one at eachmouth of the pore, and one in the region of residue S31. Theamantadine-H⁺ profile exhibits a minimum close to S31 and abarrier near residue I42. This provides a molecular model foramantadine-H⁺ block of M₂ channels. The profile for cyclopentylamine-H⁺does not exhibit such a barrier. It is predicted that cyclopentyl-amine-H⁺will not act as an M₂ channel blocker.     

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.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

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.     

Cloning and expression in E.coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor X_a, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor X_a to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli.     

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1

Denatured and reduced N-terminal extended insulin-like growthfactor-1 (AE-IGF-1) was purified from Escherichia coli extractsand subjected to in vitro folding. The renaturation processwas shown to be a function of the redox potential of the solution.Folding by different methods had no significant effect on therenaturation. A maximal yield of 60% (w/w)was obtained. Thefolded AE-IGF-1 was enzymatically converted to IGF-1. The majorby-product (20% w/w) was identified as scrambled IGF-1. Enzymaticdigestion at alkaline and acidic pH suggested two possible disulphidebond arrangements: (i) Cys⁶–Cys⁴⁷, Cys¹⁸–Cys⁶¹,Cys⁴⁸–Cys⁵²; or (if) Cys⁶–Cys⁵², Cys^l8–Cys⁶¹,Cys⁴⁷ and Cys⁴⁸ being in their reduced forms. Energy minimizationand molecular modelling suggested that the scrambled IGF-1,having reduced cysteines at positions 47 and 48, was the energeticallymost stable conformation of the two.     

1.85 A structure of anti-fluorescein 4-4-20 Fab

The crystal complex of fluorescein bound to the high-affinityanti-fluorescein 4-4-20 Fab {K_a = 10¹⁰ M^–1 at 2°C)has been determined at 1.85 Å. Isomorphous crystals oftwo isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. FunctGenet., 3, 155–160], have been grown. Both complexes crystallizewith one molecule in the asymmetric unit in space group P1,with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, = 95.15° , ß = 86.85° and = 98.01°.The final structure has an R value of 0.188 at 1.85 Åresolution. Interactions between bound fluorescein, the complementarity-determiningregions (CDRs) of the Fab and the active-site mutants of the4-4-20 single-chain Fv will be discussed. Differences were foundbetween the structure reported here and the previously reported2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins:Struct. Fund., 5, 271–280]. Our structure determinationwas based on 26 328 unique reflections — four times theamount of data used in the previous report. Differences in thetwo structures could be explained by differences in interpretingthe electron density maps at the various resolutions. The r.m.s.deviations between the variable and constant domains of thetwo structures were 0.77 and 1.54 Å, respectively. Fourregions of the light chain and four regions of the heavy chainhad r.m.s. backbone deviations of >4 Å. The most significantof these was the conformation of the light chain CDR 1.     

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).     

Molecular modeling of the amyloid-{beta}-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 1–40)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study.     

The sequence and X-ray structure of the trypsin from Fusarium oxysporum

The trypsin from Fusarium oxysporum is equally homologous totrypsins from Streptomyces griseus, Streptomyces erythraeusand to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibitedform of the enzyme has been crystallized from 1.4 M Na₂SO₄,buffered with citrate at pH 5.0–5.5. The crystals belongto space group P2₁ with cell parameters a=33.43 Å, b=67.65Å, c=39.85 Å and ß=107.6°. There isone protein molecule in the asymmetric unit. X-ray diffractiondata to a resolution of 1.8 Å were collected on film usingsynchrotron radiation. The structure was solved by molecularreplacement using models of bovine and S.griseus trypsins andrefined to an R-factor of 0.141. The overall fold is similarto other trypsins, with some insertions and deletions. Thereis no evidence of the divalent cation binding sites seen inother trypsins. The covalently bound inhibitor molecule is clearlyvisible.