Amyloid-like Properties of Bacterial Inclusion Bodies

doi:10.1016/j.jmb.2005.02.030 J. Mol. Biol. (2005) 347, 1025–1037

Amyloid-like Properties of Bacterial Inclusion Bodies

Mar Carrio1,2, Nuria Gonzalez-Montalban1,2, Andrea Vera1,2

Antonio Villaverde1,2 and Salvador Ventura1,3*

1Institut de Biotecnologia i deBiomedicina, UniversitatAutonoma de Barcelona, 08193Bellaterra (Barcelona), Spain

2Departament de Genetica i deMicrobiologia, UniversitatAutonoma de Barcelona, 08193Bellaterra (Barcelona), Spain

3Departament de Bioquimica iBiologia Molecular, UniversitatAutonoma de Barcelona, 08193Bellaterra (Barcelona), Spain

0022-2836/$ - see front matter q 2005 E

Abbreviations used: IB, inclusionprotein; FTIR, Fourier transform infrTh-T, thioflavin-T; TNS, 2-(p-toluidisulfonate.E-mail address of the correspond

[email protected]

Bacterial inclusion bodies are major bottlenecks in protein production,narrowing the spectrum of relevant polypeptides obtained by recombinantDNA. While regarded as amorphous deposits formed by passive andrather unspecific precipitation of unfolded chains, we prove here thatthey are instead organized aggregates sharing important structuraland biological features with amyloids. By using an Escherichia colib-galactosidase variant, we show that aggregation does not necessarilyrequire unfolded polypeptide chains but rather depends on specificinteractions between solvent-exposed hydrophobic stretches in partiallystructured species. In addition, purified inclusion bodies are efficient andhighly selective nucleation seeds, promoting deposition of solublehomologous but not heterologous polypeptides in a dose-dependentmanner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which,jointly with Fourier transform infra red spectroscopy data, indicates a highlevel of organized intermolecular b-sheet structure. The evidencesof amyloid-like structure of bacterial inclusion bodies, irrespective ofpotential applications in bioprocess engineering, prompts the use ofbacterial models to explore the molecular determinants of proteinaggregation by means of simple biological systems.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: inclusion bodies; protein folding; protein production; proteinmisfolding; protein aggregation
*Corresponding author
Introduction

In the last few years, protein aggregation hasemerged from a neglected area of protein chemistryas a transcendental issue in biological and medicalsciences.1 The biological relevance of proteinaggregation has been shown to be much higherthan previously realized. In this regard, an increas-ing body of evidence supports the anomalousmisassembly of proteins into insoluble amyloiddeposits as the fundamental cause behind somedebilitating human disorders of growing incidence,such as Alzheimer’s disease, type II diabetes andthe transmissible spongiform encephalopathies.2–5

No sequence or structural similarity is apparentbetween any of the diverse proteins that accumulateextracellularly in the different amyloidoses. In spite

lsevier Ltd. All rights reserve

body; TSP, tailspikea red; CR, Congo red;nylnaphthalene)-6-

ing author:

of their heterogeneity and regardless of their source,all the polypeptides involved aggregate into amyl-oid fibrils displaying similar features, namely:(1) binding to certain dye molecules such asCongo red (CR: C32H22N6Na2O6S2) or thioflavin-T(Th-T: C17H19ClN2S); (2) similar fibrillar morph-ologies; and (3) the aggregated proteins beingorganized in a cross-b-sheet architecture.6 Contraryto these structured fibrils, proteins often aggregateinside the cells into less ordered and dense proteindeposits called inclusion bodies (IBs).7 IBs occurnaturally in both prokaryotic8 and eukaryotic cells,9

but they are especially common when foreignproteins are overproduced in bacterial systems.10

In contrast to the explosion of knowledge about thefunctional and structural determinants of amyloidaggregation, the architecture and mechanisms of IBformation have been poorly studied. Despite theobvious interest of bacterial models for monitoringin vivo protein aggregation, the formation of IBs inthis type of cells has been regardedmainly as a side-effect to be minimized to enhance the productivityof bacterial cell factories.11

Interestingly, several observations suggest that

d.

1026 Amyloid Properties of Bacterial Inclusion Bodies

aggregation into IBs is much more likely to arisefrom specific and selective mechanics such as thosedriving amyloid polymerization. In this context, IBshave usually been regarded as a convenient sourceof relatively pure polypeptide, and it has beensuggested that the intermolecular interactionsdriving aggregation may occur through homo-logous protein patches.12 Other cell proteinstrapped in the aggregates are contaminants fromthe purification process,13 proteolytic fragments ofthe aggregating protein14 or other misfolding-proneproteins deposited by titration of chaperonesduring recombinant gene expression.15,16 Recently,several proteins unrelated to any known humandisease have been found to be able to organizein vitro into higher-order structures that fulfil allcharacteristics of an amyloid.17–20 Therefore, theability to form amyloids might be a genericproperty of the polypeptide backbone, the specificamino acid sequence rather determining the aggre-gation propensity of given protein species.21 Thisappears to be true for bacterial IBs as well, since awide repertoire of different, unrelated recombinantproteins form these aggregates in Escherichia coli.22

The amino acid sequence modulates protein depo-sition also in IBs, as shown in several proteinsystems in which point mutations dramaticallyaffect aggregation.23–26

In the present study, we have used a modelaggregation-prone protein, the b-galactosidasederivative VP1LAC, to generate insights into thesimilarity of the forces and structural constraintsdriving IBs and amyloid aggregation. The resultsobtained indicate clearly that protein aggregationinto bacterial IBs is a specific event based on finemolecular discrimination processes and that theresulting aggregates are more ordered than pre-viously thought, displaying clear amyloid-likeproperties.

Results

VP1LAC and b-galactosidase exhibit differentsolubility in E. coli

VP1LAC is an aggregation-prone, b-galactosidasederivative with the VP1 capsid protein of foot-and-mouth disease virus (VP1) fused at the aminoterminus of the bacterial enzyme.14 When over-produced in E. coli, VP1LAC locates in the cyto-plasm in both the soluble and insoluble cellfractions, its soluble form being enzymaticallyactive and the insoluble form aggregating asstandard IBs.27 The relative fraction of insolubleVP1LAC is slightly variable, but in a wild-typestrain and a few hours after the induction of geneexpression it can represent more than 50%.28 On thecontrary, the parental b-galactosidase (LACZ) foldsproperly and it is essentially soluble and active,the residual insoluble fraction being not higherthan 5%.28

VP1LAC aggregates in vitro from a foldedmonomeric state

To measure whether the different extent of in vivodeposition into IBs can be related to the intrinsicaggregation propensities of VP1LAC and LACZproteins, we purified the soluble forms and moni-tored their in vitro deposition. At room temperature,protein aggregates develop spontaneously in puri-fied solutions of soluble VP1LAC. The kinetics offormation of aggregates in vitro is sigmoid, exhibi-ting an initial slow phase followed by an accelera-tion of the aggregation rate and terminating at aplateau where aggregates and soluble protein existin equilibrium (Figure 1). The protein precipitatedout of the solution at the end of the reactionaccounted for about 15–20% of the total amount.As expected, LACZ protein did not aggregate under

Figure 1. Spontaneous aggrega-tion of soluble VP1LAC and LACZ.Kinetics of aggregation of initiallysoluble VP1LAC (B) and LACZ(C) solutions monitored by time-dependent increase of turbidity at350 nm.

Figure 2. Secondary structurecontent of soluble LACZ andVP1LAC. Far-UV circular dichro-ism spectra of soluble LACZ (con-tinuous line) and VP1LAC (brokenline) measured at 25 8C.

Amyloid Properties of Bacterial Inclusion Bodies 1027

the same conditions, since no significant increasein turbidity could be detected (Figure 1). Hence, thein vitro relative aggregation capabilities of VP1LACand LACZ resemble those found in vivo.

Partially unfolded proteins exhibit much higheraggregation propensities than their native counter-parts.19 Soluble VP1LAC solutions display detect-able enzymatic activity,28 showing that the proteincan adopt the b-galactosidase crude fold. However,it cannot be discarded that the fusion of VP1 at its Nterminus could promote significant changes in itsoverall structure, which might account for theobserved differences in aggregation propensities.We used far-UV circular dichroism (CD) to analysethe secondary structure content of both proteins.The CD spectrum indicated that LACZ is domi-nated by b-sheet (42%), as seen by the characteristic

minimum at 215 nm in Figure 2, with lesseramounts of a-helix (7%). VP1LAC CD spectrumresembled that of LACZ, with small changes inshape and magnitude (Figure 2), and correspondedto a folded protein with similar estimated b-sheet(41%) and a-helix (8%) content. Thus, the aggrega-tion properties of VP1LAC cannot be explained asresulting from unspecific precipitation of an exten-sively unfolded protein chain.To further investigate the conformational state of

VP1LAC, we used the polarity-sensitive fluorescentprobe 2-(p-toluidinylnaphthalene)-6-sulfonate(TNS). TNS binds to exposed hydrophobic sur-faces,29 resulting in an increase in fluorescenceemission compared with the emission of free TNS inaqueous solution. Little TNS binding to LACZ wasdetected, while the presence of VP1LAC in the

Figure 3. Conformation of sol-uble LACZ and VP1LAC. Fluor-escence emission spectra of TNSincubated in the presence of sol-uble LACZ (C) or VP1LAC (B).


solution led to a more than fourfold increase influorescence emission (Figure 3), indicating theexposure of some hydrophobic clusters hidden inthe native protein.

The functional LACZ is a homotetrameric pro-tein, in which each identical monomer foldsindependently to further dimerize. Finally, dimersassociate to form the functional tetramer.30 Oneexplanation for the higher TNS binding capacityand aggregation propensity of VP1LAC could be anaberrant quaternary structure. To compare theoligomeric states of VP1LAC and LACZ, weanalysed both samples by non-denaturing gelelectrophoresis. As shown in Figure 4, only asmall amount of VP1LAC is able to ensemble intotetramers and most of the protein remains asmonomers. In addition, we observed the assemblyof VP1LAC into high molecular mass polymericspecies, completely absent from the LACZ solution.Monomeric VP1LAC might expose at least part ofthe polypeptide main chain and hydrophobic side-chains that are otherwise buried in the overall foldof the native protein. These polypeptidic regions arecapable of strong intermolecular interactions andmay well account for the increased aggregationpropensity.

Protein aggregation as IBs is a selective process

In vivo, IBs are though to be formed by the growthof a limited number of “founder” aggregates, whichdrive further the aggregation process. While this is a

Figure 4.Oligomeric state of LACZ and VP1LAC. Non-denaturing gel electrophoresis of heat-denatured LACZ(lane 1), native LACZ (lane 2) and VP1LAC (lane 3). O,oligomer; T, tetramer; M, monomer.

plausible explanation for the reduced number of IBs(usually one) formed in the cell cytoplasm,27 to thebest of our knowledge there is no experimentalevidence that IBs can act as nuclei of aggregationprocesses. To test if this occurs in vitro, we seededsoluble VP1LAC with 1/10 (w/w) of purifiedVP1LAC IBs, and monitored the time-course ofaggregation by measuring light-scattering (Figure5). Soluble VP1LAC did not aggregate significantlythroughout the experiment. However, the presenceof preformed VP1LAC IBs induced its immediateincorporation into the insoluble fraction following afirst-order kinetics. The aggregation was completeafter five hours and the fraction of protein pre-cipitated out of the solution was higher than 40%(data not shown). In contrast, the presence of (1/10)VP1LAC IBs was unable to seed the aggregationof the sequentially/structurally related proteinLACZ (Figure 5(a)), indicating that a certain degreeof specificity is required for the aggregation tooccur. To distinguish whether the capability ofVP1LAC to be seeded by IBs is indeed a selectiveprocess driven by proper interactions between thesoluble protein and its IB version, or on the contrary,it is just reflecting its higher intrinsic propensity toaggregate, we tried to seed soluble VP1LAC with1/10 (w/w) of preformed IBs of three unrelatedpolypeptides; the tailspike protein (TSP) of thebacteriophage P22, a mutant of a-spectrin SH3domain (SPC-PI3DT) and the human immuno-deficiency virus type 1 (HIV-1)-encoded protease.TSP is a homotrimeric protein that undergoes acomplex folding pathway from which unfoldedintermediates collapse as IBs under overproductionconditions.31 SPC-PI3DT is a mutant of the highlysoluble SH3 domain of a-spectrin engineered toform amyloid fibrils in vitro,20 and expressedexclusively as IBs in E. coli. The HIV-1 proteasehydrolyses HIV polyproteins into functional pro-teins that are essential for virus assembly and it isdeposited as IBs when over-expressed in bacteria.32

Surprisingly, TSP, SPC-PI3DT and HIV-1 proteaseIBs were unable to seed significantly the aggrega-tion of soluble VP1LAC, and only slight linearincreases in turbidity occurred with time (Figure5(a)). These results demonstrated clearly thatVP1LAC deposition into IBs is a selective process.

To explore if there is a dose-response relationshipbetween seeding and aggregation levels in thisparticular system, we incubated soluble VP1LACwith increasing concentrations of VP1LAC IBs. Asshown in Figure 5(b), the rate of aggregation in theassay is highly dependent on the initial concen-tration of IBs that act as seeds. Concentrations of IBsbelow 5% have detectable but small effects onsoluble VP1LAC aggregation rates. IBs at a concen-tration of 5% reduce the lag time of the reactionsignificantly, whereas a 10% IBs eliminate this phasecompletely, resulting in fast aggregation of solubleprotein.

We wondered whether the in vitro formedaggregates recovered from the seeded reactionwere able to act as new seeds. To test this, we

Figure 5. Seeding of solubleVP1LAC by homologous IBs.Kinetics of aggregation monitoredby time-dependent increase ofturbidity at 350 nm. (a) SolubleVP1LAC incubated with (1/10)VP1LAC IBs is shown in red,soluble VP1LAC incubated with(1/10) TSP IBs is shown in blue,soluble VP1LAC incubated with(1/10) SPC-PI3DT IBs is shown ingreen and soluble VP1LAC incu-bated with (1/10) HIVP IBs isshown in purple. Soluble LACZis shown in black. Finally, solubleLACZ incubated with (1/10)VP1LAC IBs is represented inorange. (b) Soluble VP1LACseeded with increasing concen-trations of VP1LAC IBS: red, 0%;green, 1%; purple, 2.5%; blue, 5%;and black, 10%.


seeded soluble VP1LAC with 1/10 (w/w) ofpreformed VP1LAC IBs and collected the insolublefraction at the end of the aggregation reaction. Next,we incubated fresh soluble VP1LAC with 1/20(w/w) of the recovered aggregates and followed thetime-course of aggregation by monitoring turbidity.As shown in Figure 6, the in vitro recoveredaggregates were able to accelerate the incorporationof new soluble VP1LAC into the aggregatedfraction.

It has been argued that aggregates formed in vitroresemble those formed in vivo.33 Thus, it makessense to determine if the protein precipitated out ofthe solution in an unseeded reaction is able to act asa specific nucleation agent. We recovered thespontaneously precipitated VP1LAC from anunseeded reaction after 48 hours and incubatedwith fresh soluble VP1LAC in a ratio 1/20 (w/w).Interestingly enough, the turbidity of the solution

increased rapidly, indicating the formation of newaggregates and therefore seeding (Figure 6).To better characterize the different VP1LAC

aggregates generated in this study, we usedtransmission electron microscopy (TEM) to exploretheir morphology. Figure 7 shows representativemicrographs of the various samples. VP1LAC IBsdisplay the typical electro-dense ellipsoidal shape.Few intact VP1LAC IBs could be detected in theaggregate material coming from an IB seededsolution. In contrast, in these preparations, electro-dense material was usually surrounded by lessdense amorphous aggregates. This observationmaysuggest direct incorporation of incoming materialon top of pre-existing IBs, but further studies wouldbe required to confirm this interpretation. Spon-taneously in vitro aggregated VP1LAC exhibiteddifferent shapes ranging from globular densestructures to annular ones, all them relatively

Figure 6. Seeding of solubleVP1LAC by homologous in vitroaggregates. Kinetics of aggregationmonitored by time-dependentincrease of turbidity at 350 nm.Soluble VP1LAC incubated in theabsence (no symbols), or in thepresence of (1/20) unseededVP1LAC aggregates (B) or(1/20) previously seeded aggre-gates (C) is shown.


small in comparison to IBs. When this material wasused to seed a soluble VP1LAC solution (Figure 6),in general, the formation of bigger electro-denseaggregates was in general observed.

IBs are ordered aggregates

Bacterial IBs have often been regarded as dis-ordered precipitates of non-specifically coagulatedpolypeptide chains. Nevertheless, the resultsshown above suggest that aggregation is the resultof specific interactions between aggregation-pronespecies. Therefore, IBs should posses a detectabledegree of intrinsic organization. To explore thispossibility, we used Fourier transform infra red(FTIR) spectroscopy, among the few techniquessuitable for obtaining structural information ofaggregated proteins, to compare the secondarystructure content of soluble and IBs forms ofVP1LAC. In functional b-galactosidase, each mono-mer folds into five sequential domains, four of themwith a b-sandwich fold and one with a–b barrelarchitecture.34 According to this, the FTIR spectrumof soluble VP1LAC in the amide region I wasdominated by a peak at 1634 cmK1 that arisesfrom intramolecular C]O vibrations in b-sheets(Figure 8(a)). Minor peaks were distinguishedat 1644 cmK1, 1654 cmK1, and 1676 cmK1, usuallyassigned to random, a-helix and antiparallel b-sheetconformations, respectively. The FTIR of VP1LACIBs was completely different, and a strong sharppeak was detected at 1621 cmK1. Thisband, together with the presence of a band at1691 cmK1, is indicative of newly formed, extendedantiparallel pleated b-sheet structures in the aggre-gates. The presence of these two bands is indeedused as a diagnostic tool in most protein amyloid

fibrils. The band located at 1651 cmK1 has beenattributed in several models to the presence ofdisordered structures.35 In order to asses if theapparition of FTIR bands coincident with those inthe spectrum of amyloidal structures is a particularfeature of VP1LAC IBs or a general property of thiskind of aggregates, we analysed two additional IBmodels. For that, we selected the closely relatedprotein LACVP1, in which the alignment of the twofusion partners is inverted with respect toVP1LAC,14 and the above-mentioned TSP, sequen-tially and structurally unrelated to VP1LAC. Asshown in Figure 8(b), the FTIR spectra of these threedifferent IBs were very similar, all of them sharingthe bands associated with the formation of inter-molecular b-sheets.

Amyloid-specific dyes bind to bacterial IBs

The presence of polypeptidic chains in a crossedb-pleated sheet conformation is a testable propertyof amyloid fibrils. Then, Th-T fluorescence has beendescribed as a specific marker for the extendedsheet conformation of amyloid structure. When thisdye binds to amyloid fibrils there is a largeenhancement in the fluorescence of Th-T relativeto free dye.36 As it appeared from our results thatIBs possess a quite homogeneous structure in whichintermolecular extended b-sheet is predominant,we tested their Th-T binding ability. Figure 9(a)shows the fluorescence spectra of Th-T incubated inthe presence of soluble VPILAC or different IBs. Itcan be observed that, whereas soluble proteinexhibited little binding, a sixfold increase in thefluorescence emission maximum of Th-T occurredafter binding to VP1LAC IBs. In accordance withthe similar global structure displayed by different

Figure 7. Micrographs of VP1LAC aggregates. (a) Electron micrographs of VP1LAC IBs. In (b) and in (c) solubleVP1LAC seeded with 10% VP1LAC IBs, arrows indicating amorphous aggregates on top of ellipsoidal electron-denseones. In (d) and in (e) unseeded in vitro VP1LAC aggregates. In (f) soluble VP1LAC seeded with 5% unseeded in vitroVP1LAC aggregates. The scale bar represents 500 nm.


IBs, LACVP1 and TSP IBs also promoted afluorescence emission enhancement.

The conformational features of IBs were furtherinvestigated bymeasuring the binding to CR, whichhas been suggested to bind most amyloids throughtheir b-pleated fibrillar structure. CR bindingappears to occur by a combination of both specifichydrophobic and electrostatic interactions. Alone, it

exhibits an absorbance maximum at 490 nm thatshifts to red once it binds amyloid material.37 Figure9(b) shows the absorption spectra of CR incubatedin the presence of soluble VPILAC or different IBs.No CR binding was detected to soluble VP1LAC,while the presence of VP1LAC, LACVP1 or TSP IBspromoted a strong shift of the absorbance maxi-mum from 491 nm to 505 nm, as described for

Figure 8. Structural properties ofIBs. (a) FTIR spectrum of solubleVP1LAC (broken line) andVP1LAC IBs (continuous line).The positions of the spectral com-ponents are estimated from thesecond derivative analysis.(b) FTIR spectrum of VP1LAC IBs(continuous line), LACVP1 IBs(broken line) and TSP IBs (dottedline).


amyloids.37 Overall, the tintorial properties of IBsconfirm that they share some common structuralfeatures with amyloid fibrils.

Discussion

Protein aggregation as insoluble bacterial IBs is acommon obstacle in bioengineering. Protein aggre-gation occurs also in eukaryotic cells and in somecases it is linked intimately to strongly disabilitatinghuman disorders like Parkinson’s disease. Asidefrom these particular cases, little attention has beenpaid to the mechanism of protein aggregation intoIBs, and most of the efforts within protein misfold-ing research have been devoted to study protein

aggregation as amyloid fibrils. The formation ofsuch fibrils is a highly specific process, driven byparticular intermolecular interactions between theaggregating polypeptidic chains. Evidence of thisselectivity is found in the seeding behaviour of mostamyloidogenic proteins. In contrast, bacterial IBshave usually been seen as disordered aggregates,their formation being driven by non-specific inter-actions between aberrantly exposed hydrophobicsurfaces. However, the structural comparativeanalysis of folding intermediates and IB proteinhas prompted us to suggest a relationship betweenaggregation in vitro and in vivo.33 In addition,protein aggregation in vitro has been shown to behighly specific.38 Among others, these observationsgenerated strong suspicions about the specificity of

Figure 9. Binding of amyloiddyes to IBs. (a) Fluorescenceemission spectra of Th-T in thepresence of soluble VP1LAC(black), VP1LAC IBs (green),LACVP1 IBs (red) and TSP IBs(blue). (b) Absorption spectra ofCongo red in the presence ofsoluble VP1LAC (black), VP1LACIBs (green), LACVP1 IBs (red) andTSP IBs (blue).


IB formation and its relationship with amyloid-genesis,39 although the biological extent of thisanalogy has not been tested further. The resultspresented here demonstrate that in vivo proteinaggregation into bacterial IBs is in fact a veryselective reaction mechanistically similar to amyl-oid formation, and provide a rationale to under-stand this process.

Amyloid formation is proposed to proceed by amechanism similar to crystallization, where thegrowth of aggregates requires the formation of anucleus, the highest-energy species on the pathway.Once a nucleus has formed, the subsequentaddition of monomers to the growing polymerbecomes energetically favourable, and growthproceeds rapidly. This model has been proved

experimentally for several amyloid-forming pro-teins, including Alzheimer’s peptide,40 a-synu-clein41 and prions.42 According to our results, thisnucleation-dependent aggregation model is validfor bacterial IB formation too. Here, we demon-strate, for the first time, that IBs can seed specificallythe aggregation of soluble protein in a dose-dependent manner. In this way, the presence ofpre-formed VP1LAC IBs strongly promotes incor-poration of soluble VP1LAC into the aggregatedfraction, reducing or eliminating the lag phase,suggesting the smooth incorporation of monomersinto IBs without a high-energy barrier. In addition,the preliminary morphological data in this studysuggest that IBs may incorporate incoming mono-mers directly onto their surface. Overall, the


seeding ability of IBs explains the reduced numberof this aggregates usually found in the bacterialcytoplasm.

The recruitment of soluble VP1LAC into IBsdepends on specific interactions between certainregions exposed both in IBs and the solublecounterpart, and it cannot be attributed merely tomolecular crowding or unspecific hydrophobicinteractions. In this way, while both in vivo andin vitro preformed VP1LAC aggregates seed solubleVP1LAC aggregation, unrelated IBs do not seed thereaction specifically. In addition, VP1LAC IBs haveno effect on soluble LACZ, indicating that theregion promoting aggregation in VP1LAC is notpresent or not exposed in the native form. This canbe related to the experimental evidence thatVP1LAC displays solvent-exposed hydrophobicsurfaces absent from LACZ. A simple explanationfor this behaviour could be that specific interactionsbetween the N-terminal VP1 peptide mediate theincorporation of soluble VP1LAC into homologousIBs. However, VP1LAC fusions in which the VP1fragment appeared partially proteolysed stillaggregate, disfavouring this hypothesis (N.G.-M.,unpublished results). VP1LAC and LACZ displayquite similar global secondary structure, but themutant form is not able to gain quaternarystructure. Hence, it is feasible that the stickyregion(s) is(are) located in the LACZ moiety buthidden from solvent in the tetrameric state. Gainingthe native conformation in multidomain proteinsinvolves rather independent folding of the distinctdomains followed by pairing and packing reactionsbetween them.43 The data suggest that the differentdomains within the VP1LAC monomer do notrearrange properly, precluding tetramerization andfavouring aggregation by incorrect intermolecularassociation of the secondary structure elements ofdifferent polypeptide chains. In this sense, theaggregation of VP1LAC would resemble amyloidfibril formation by transthyretin, in which dis-sociation of the tetrameric structure is a prerequisitefor aggregation to occur.44

The need for purity and quantity has forcedresearchers to obtain polypeptides for amyloidstructural studies from recombinant bacteria. Inter-estingly, proteins able to form amyloid fibrils invitro, are usually produced in E. coli as IBs.45–47

Consistently, mutants of these proteins withreduced amyloid propensity are expressed inE. coli as more soluble proteins,48 whereas givinga previously soluble protein increased amyloidpropensity results in accumulation as IBs.49,50

Moreover, when amyloid proteins have beendesigned de novo, all proteins displaying amyloidproperties in vitro accumulated in vivo as bacterialIBs.51 On the other hand, the rational introductionof mutations that convert these aggregation-proneproteins into monomeric b-sheet forms resulted inthe expression of soluble protein.52 Hence, theseobservations suggest strongly that the inter-molecular interactions that drive proteins to asso-ciate in vitro as fibrils also favour aggregation as IBs

in vivo, and that both phenomena depend on thetendency to self-aggregation associated with par-ticular protein sequences.

Protein association as IBs, being a specificprocess, should render some degree of internalorder in the final aggregate, as it occurs in amyloidfibrils. Our data show that IBs, contrary to theconventional view, are highly ordered proteinaggregates, the different polypeptidic chainsbeing condensed in an extended b-sheet manner,as demonstrated by FTIR. These structures areprobably stabilised by hydrogen bonds betweendifferent chains resulting in intermolecularb-sheets. Our results are highly coincident withthose previously reported by Fink and co-workers,who examined the secondary structure in variousaggregate forms, including inclusion bodies fromdifferent proteins.33,53 In all cases, a large amount ofnew b-sheet structure, compared with that inthe native state, was observed. Georgiou andco-workers reported an increase in b-sheet structureand a significant decrease in a-helical content inb-lactamase IBs.54 More recently, the presence ofthe FTIR band associated with the formation of newb-sheet structure has been used as a diagnostic ofthe level of protein deposition in vivo in bacteriaduring heterologous protein expression.55 Despitethe fact that the amount of secondary and dis-ordered structure in IBs may vary from one proteinto another, it can be concluded that proteinconformation in IBs clearly differs from that in thenative state. In particular, the major FTIR bands inthe amide I region observed for the IBs analysed inthis study suggest that its organization closelyresembles that of amyloid fibrils, although in IBs afraction of the polypeptidic chain/s appears to beunstructured. As it happens in amyloid fibrils, thenewly formed b-sheets in IBs are tightly packed inanti-parallel orientation with short hydrogen bondsstabilizing them, as deduced from bands around1621 cmK1 and 1690 cmK1. Furthermore, we haveconfirmed the resemblance of the internal organi-zation of both kinds of aggregates by showing thatthe so-called amyloid-diagnostic dyes also bind toIBs. This could indicate that these dyes recognizethe crossed b-pleated sheet conformation in theaggregate rather than its supramolecular structure.Other experimental data support this view.56,57

Overall, we have shown that the study ofbacterial systems should be seriously consideredwhen exploring protein misfolding and aggrega-tion, since they can provide new clues about thestructural/sequential constraints underlyingprotein deposition by means of a fast, simple andbiologically relevant experimental model.

Methods

Protein and IB purification

Protein VP1LAC and its parental LACZ were producedin E. coliMC4100 in rich medium under the control of the


strong lambda lytic promoters.27 The purification ofsoluble forms was performed from crude cell extractsobtained by ultrasonication, by single-step affinity Sepha-rose chromatography using the non-hydrolysable sub-strate of b-galactosidase TPEG as described.58 VP1LACIBs were purified from cell extracts five hours after theinduction of gene expression, by detergent-based pro-cedures as described.59 Purified protein concentration ineither soluble or as IBs was quantified by the Bradfordassay after denaturation (8 M urea) using commercialb-galactosidase as an internal standard. Non-denaturingPAGE (7.5% (w/v) acrylamide, pH 8.3) was used toinvestigate the oligomeric status of the soluble forms.Bands were immunodetected byWestern blot by using ananti-b-galactosidase rabbit serum and a secondary,horseradish peroxidase-labelled antibody.

Quantification of protein deposition and aggregationkinetics

Protein deposition was analysed by centrifuging 1 mlaliquots of protein samples, at 30,000g for one hour at theend of the experiment. The fraction of aggregated proteinwas calculated as the ratio between the amount of proteinremaining in the pellet and the amount of protein in thesupernatant. The experiments were carried out intriplicate to minimise errors during sample handling.Protein absorbance at 280 nm and turbidity at 350 nmwere measured on a CARY-100 Varian spectrophotometerat different times during the VP1LAC unseeded aggrega-tion experiment. The initial concentration of the proteinused was 100 mg/ml.The kinetics of aggregation in seeding experiments

were followed by continuously monitoring the absorb-ance at 350 nm during 300 minutes at 25 8C. Total proteinconcentration in the assay was 100 mg/ml. Stirring wasmantained during all the experiment to avoid aggregatedeposition. Soluble protein fractions were ultracentri-fuged inmediately before the experiment to avoid thepresence of artifactual aggregates at the beginning ofthe assay. To initiate seeding of soluble protein by IBs,10–100 ml of IBs at 100 mg/ml were added to 900 ml ofsoluble protein at the same concentration. In all cases, thecontribution to turbidity of IBs alone was measured inparallel with the seeding experiment and subtracted fromthe corresponding spectra.

Conformational studies

Circular dichroism spectra in the far-UV region wereobtained by using a Jasco-710 spectropolarimeter at 25 8C.Spectra were recorded for soluble proteins (10 mg/ml) in50 mM sodium phosphate (pH 7.0). Ten accumulationswere averaged to generate each spectrum. Proteinsecondary structure content was calculated using theCDPro suite.†The fluorescence emission spectra of TNS with and

without soluble protein were recorded at 25 8C in aPerkin–Elmer 650–40 fluorescence spectrophotometer.Protein samples (100 mg/ml) were diluted tenfold intothe corresponding buffer containing 100 mM TNS.Samples were equilibrated for five minutes at 25 8Cbefore acquiring fluorescence spectra. The fluorescenceof TNS was excited at 323 nm and the emission measured

† http://lamar.colostate.edu/~sreeram/CDPro/main.html

between 350 nm and 600 nm. Both excitation and emis-sion slits were set at 10 nm.For FTIR spectroscopy analysis, IBs were dried for one

hour in a Speed-Vac system before analysis to reducewater interference in the infrared spectra. The structure ofthe dry aggregates was analysed directly in a BrukerTensor FTIR spectrometer. The FTIR spectrum of thenative protein was acquired after air-drying the proteinsolution. For each spectrum, 20 interferograms werecollected and averaged. All processing procedures werecarried out so as to optimize the quality of the spectrum inthe amide I region, between 1550 cmK1 and 1700 cmK1.Second derivatives of the amide I band spectra were usedto determine the frequencies at which the differentspectral components were located.

Transmission electron microscopy

A 5–10 ml aliquot of different VP1LAC aggregates wasplaced on carbon-coated copper grids, and left for fiveminutes. The grids were then washed with distilled waterand stained with 2% (w/v) uranyl acetate for another twominutes before analysis using a HitachiH-7000 trans-mission electron microscope operating at acceleratingvoltage of 75 kV.

Binding to amyloid-diagnostic dyes

Samples were tested for CR binding by the spectro-scopic band-shift assay as described.35 Either solubleprotein or IBs were diluted in reaction solution (5 mMsodium phosphate (pH 7.0), 150 mM NaCl) containing5 mM CR. Samples were equilibrated for five minutes at25 8C before analysis. Absorption spectra were collectedtogether with negative control solutions of dye in theabsence of protein and of protein samples in theabsence of dye, subtracting the scattering contributionfrom the samples spectra, on a CARY-100 Varianspectrophotometer.Thi-T binding assays were carried out using aliquots of

100 ml drawn from 100 mg/ml soluble or IBs proteinsamples. These aliquots were diluted into buffer (10 mMsodium phosphate (pH 7.0), 150 mM NaCl,) containing65 mM Th-T, and adjusted to a final volume of 1 ml.Fluorescence data were collected after five minutes toensure that thermal equilibrium had been achieved.Fluorescence emission spectra were recorded using anexcitation wavelength fixed at 440 nm on a Perkin–Elmer650–40 fluorescence spectrophotometer. Apertures of10 nm were fixed in both excitation and emission slits.

Acknowledgements

This work has been supported by BIO2004-00700from MEC, Spain and 2002SGR-0099 (AGAUR).S.V. is supported by a “Ramon y Cajal” projectawarded by the MCYT and co-financed by theUniversitat Autonoma de Barcelona. We thank DrJosep Vendrell for revising this manuscript andFrancesc X. Aviles for lab facilities.

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Edited by C. R. Matthews

(Received 26 October 2004; received in revised form 10 February 2005; accepted 14 February 2005)

Amyloid-like Properties of Bacterial Inclusion Bodies

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