In vivo copper- and cadmium-binding ability of mammalian metallothionein domain

The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of Cu–ßMTand Cd–ßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn₃–ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

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.     

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-gal–proAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor X_a. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-gal–proAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor X_a digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorX_a, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDS–polyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex

Molecular recognition and site-directed mutagenesis are usedin combination to identify kinetically, transition state interactionsbetween glucoamylase (GA) and the substrate maltose. Earlierstudies of mutant Glu180 – Gin GA had indicated a rolein substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly,P.J. and Svensson, B. (1990) Protein Engng, 3, 193–198).Here, changes in activation energies calculated from measuredk_cat/K_m values for a series of deoxygenated maltose analoguesindicate hydrogen bonding between the mutant enzyme and the3-OH group of the reducing end sugar ring. Using the same substrateanalogues and determining activation energies with wild-typeGA an additional hydrogen bond with the 2-OH group of maltoseis attributed to an interaction with the carboxylate Glu180.This novel combination of molecular recognition and site-directedmutagenesis enables an enzyme substrate transition state contactto be identified and characterized even without access to thethree dimensional structure of the enzyme. Given the distantstructural relationships between glucoamylases and several starchhydrolases (Svensson, B. (1988) FEBS Lett., 230, 72–76),such identified contacts may ultimately guide tailoring of theactivity of these related enzymes.     

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.     

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 structure of a designed peptidomimetic inhibitor complex of {alpha}-thrombin

Thrombin displays remarkable specificity, effecting the removalof fibrinopeptides A and B of fibrinogen through the selectivecleavage of two Arg–Gly bonds between the 181 Arg/Lys–Xaabonds in fibrinogen. Significant advances have been made inrecent years towards understanding the origin of the specificityof cleavage of the Argl6–Gly17 bond of the A-chain ofhuman fibrinogen. We have previously proposed a model for thebound structure of fibrinopeptide A_7–16 (FPA), based uponNMR data, computer-assisted molecular modeling and the synthesisand study of peptidomimetic substrates and inhibitors of thrombin.We now report the structure of the ternary complex of an FPAmimetic (FPAM), hirugen and thrombin at 2.5 Å resolution(R-factor = 0.138) and specificity data for the inhibition ofthrombin and related trypsin-like proteinases by FPAM. The crystallographicstructures of FPA and its chloromethyl ketone derivative boundto thrombin were determined. Although there are differencesbetween these structures in the above modeled FPA structureand that of the crystal structure of FPAM bound to thrombin,the , angles in the critical region of P1–P2–P3in all of the structures are similar to those of bovine pancreatictrypsin inhibitor (BPTI) in the BPTI–trypsin complex andD–Phe–Pro–Arg (PPACK) in the PPACK–thrombinstructure. A comparison between these and an NMR-derived structureis carried out and discussed.     

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.     

A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs

Using a series of homologous calcium-binding proteins, a quantitativestructure–activity relationship (QSAR), log(1/K_d) = –18.986– 1.6278(X₁) + 0.7981(X₂) + 0.2312(X₃), has been established,which relates the calcium-binding affinities (1/K_d) of the regulatoryproteins with (i) the net ligand charge (X₁) of the two calciumbinding loops, (ii) the hydrophobicity (X₂) of the ß-sheetsegment of the loops and (iii) the hydrophobicity (X₃) of thefour ‘EF-hand’ helices. It is found that the bindingaffinities are influenced by the ‘EF-hand’ pairrather than the individual ‘EF-hands’. The QSAR,in addition to explaining satisfactorily the large variationin the observed calcium affinities, can predict the affinitiesof the ‘EF-hand’ pairs in other proteins from theamino acid sequence and can also account for the changes inthe affinities caused by substitution in the hydrophobic and/ormetal-coordinating residues. Thus, this relationship can beemployed in protein design and engineering. The method is potentiallyuseful in the development of similar relationships for the bindingof other proteins to substrates, inhibitors, drugs and co-factors.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

Making tissue-type plasminogen activator more fibrin specific

The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296–299or DDD 364–366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466–470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe k_cat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296–299, where the presenceof fibrin manifests significant increases in both k_cat and K_m.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

Detection of non-topological motifs in protein structures

We present an efficient technique for the comparison of proteinstructures. The algorithm uses a vector representation of thesecondary structure elements and searches for spatial configurationsof secondary structure elements in proteins. In such recurringprotein folds, the order of the secondary structure elementsin the protein chains is disregarded. The method is based onthe geometric hashing paradigm and implements approaches originatingin computer vision. It represents and matches the secondarystructure element vectors in a 3-D translation and rotationinvariant manner. The matching of a pair of proteins takes onaverage under 3 s on a Silicon Graphics Indigo2 workstation,allowing extensive all-against-all comparisons of the data setof non-redundant protein structures. Here we have carried outsuch a comparison for a data set of over 500 protein molecules.The detection of recurring topological and non-topological,secondary structure element order-independent protein foldsmay provide further insight into evolution. Moreover, as theserecurring folding units are likely to be conformationalHy favourable,the availability of a data set of such topological motifs canserve as a rich input for threading routines. Below, we describethis rapid technique and the results it has obtained. Whilesome of the obtained matches conserve the order of the secondarystructure elements, others are entirely order independent. Asan example, we focus on the results obtained for Che Y, a signaltransduction protein, and on the profilin-ß-actincomplex. The Che Y molecule is composed of a five-stranded,parallel ß-sheet flanked by five helices. Here weshow its similarity with the Escherichia coli elongation factor,with L-arabinose binding protein, with haloalkane dehalogenaseand with adenylate kinase. The profilin–ß-actincontains an antiparallel ß-pleated sheet with -helicaltermini. Its similarities to lipase, fructose disphosphataseand ß-lactamase are displayed.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilateisomerase:indole-3-glycerol-phosphate synthase from Escherichiacoli has been crystallized, and the structure has been solvedat 4 Å resolution. Two closely related crystal forms grownfrom ammonium sulphate diffract to 2 Å resolution. Oneform (space group R32, a = 163 Å, = 29.5°) containsthe unliganded synthase domain; the second crystal form (spacegroup P6₃22, a = 144 Å, c = 158 Å) is co-crystallizedwith the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate.The structure of the synthase–inhibitor complex has beensolved by the molecular replacement method. This achievementrepresents the first successful use of a (ß)_g-barrelmonomer as a trial model. The recombinant synthase domain associatesas a trimer in the crystal, the molecules being related by apseudo-crystallographic triad. The interface contacts betweenthe three domains are mediated by those residues that are alsoinvolved in the domain interface of the bifunctional enzyme.This system provides a model for an interface which is usedin both intermolecular and intramolecular domain contacts.     

The 2 A crystal structure of subtilisin E with PMSF inhibitor

Using enzyme prepared by the DNA recombination technique, subtilisinE from Bacillus subtilis was crystallizedin space group P2₁2₁2₁with two molecules in an asymmetric unit. The crystal structureof PMSF-inhibited subtilisin E was solved by molecular replacementfollowed by refinement with the X-PLOR program. This resultedin the 2.0 Å structure of subtilisin E with an R-factorof 0.191 for 8–2 Å data and r.m.s. deviations fromideal values of 0.021 Å and 2.294° for bond lengthsand bond angles respectively. The PMSF group covalently boundto Ser221 appeared very clearly in the electron density map.Except for the active site disturbed by PMSF binding, the structuralfeatures of subtilisin E are almost the same as in other subtilisins.The calcium-binding sites are different in detail in the twoindependent molecules of subtilisin E. Based on the structure,the remarkably enhanced heat stability of mutant N118S of subtilisinE is discussed. It is very likely that there is an additionalwater molecule in the mutant structure, which is hydrogen bondedto side chains of Serll8 and its neighbouring residues Lys27and Asp 120.     

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.     

Free energy simulations of the HyHEL-10/HEL antibody-antigen complex

Free energy simulations are reported for the N31^L-D mutation,both in the HyHEL-10-HEL antibody-lysozyme complex and in theunliganded antibody, using the thermo-dynamic-cycle perturbationmethod. The present study suggests that the mutation would changethe free energy of binding of the complex by –5.6 kcal/mol(unrestrained free energy simulations), by –0.5 kcal/mol(free energy simulations with a restrained backbone) and by1.8 kcal/ mol (Poisson-Boltzmann calculations, which also usea restrained geometry model). A detailed structural analysishelps in estimating the contributions from various residuesand regions of the system. Enhanced recognition of HEL by themutant HyHEL-10 would arise from the combination of thermodynamicallymore favorable conformational changes of the CDR loops uponassociation and subsequent charge pairing with Lys96 in theantigen.     

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.