mouse hsp25, a small heat shock protein

Eur. J. Biochem. 267, 1923±1932 (2000) q FEBS 2000

Mouse Hsp25, a small heat shock proteinThe role of its C-terminal extension in oligomerization and chaperone action

Robyn A. Lindner1, John A. Carver1, Monika Ehrnsperger2, Johannes Buchner2, Gennaro Esposito3,Joachim Behlke4, Gudrun Lutsch4, Alexey Kotlyarov4 and Matthias Gaestel4,5

1Department of Chemistry, University of Wollongong, Australia; 2Institut fuÈr Biophysik und Physikalische, Biochemie,

UniversitaÈt Regensburg, Germany; 3Dipartimento di Scienze e Tecnologie Biomediche, UniversitaÁ degli Studi di Udine, Italy;4Max-DelbruÈck-Centrum fuÈr Molekulare Medizin, Berlin, Germany; 5Innovationskolleg, Zellspezialisierung, Martin-Luther-UniversitaÈt,

Halle, Germany

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and

prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short,

polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been

constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant

and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have

very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that

of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize a-lactalbumin

against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant

protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature.1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra

C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced

a-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility

during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian

sHsps is required for full chaperone activity.

Keywords: flexibility; Hsp25; molecular chaperone; small heat shock protein; spectroscopy.

The small heat shock proteins (sHsps) are a class of proteinsthat are involved in stabilizing other proteins under conditionsof stress, e.g. heat, heavy metals, oxidation (reviewed in [1,2]).As an example, many proteins will aggregate and precipitateout of solution when heated but, in the presence of sHsps, thiscan be prevented via formation of a soluble complex betweenthe sHsp and its substrate protein [3]. The sHsps are thereforeclassified as chaperone proteins, although they do not have theability to refold the substrate proteins to a significant degree,except in the presence of another chaperone protein, e.g. Hsp70[4]. The nature of the chaperone action of sHsps and itsmechanism, however, are poorly understood.

The sHsps range in subunit molecular mass from around13±42 kDa but, in the main, they are found as large oligomericspecies ranging from 200 to 1000 kDa [1,2,5,6]. sHsps sharesequence similarity in their C-terminal region and also in ashort hydrophobic phenylalanine region near their N terminus[2]. The recent determination of the crystal structure of

Methanococcus jannaschii Hsp16.5 [7], a homogeneous24-subunit sHsp aggregate from a hyperthermophilic archaeon,has shown that the conserved C-terminal region comprises awell-structured, mainly b-pleated sheet domain with manyinter-subunit interactions within the oligomer. The overallstructure is a hollow sphere with an external diameter of< 120 AÊ and an internal diameter of < 65 AÊ , the surface ofwhich is punctuated with 14 holes or `windows'.

Unlike M. jannaschii Hsp16.5, the mouse sHsp, Hsp25 doesnot form oligomers of well-defined stoichiometry but exists as aheterogeneous aggregate. Similarly, a-crystallin (the major lensprotein and also a member of the sHsp family) exists as aheterogeneous aggregate with an average mass of < 800 kDa.a-Crystallin consists of two related subunits, A and B. Genetargeting of aA-crystallin has demonstrated clearly the role ofthis protein in preventing protein precipitation and cataractformation [8]. aB-Crystallin has also been found in manynonlens tissues where it, along with other sHsps (e.g. Hsp25)act to stabilize other proteins under conditions of stress [1,2].

In previous studies, we have identified by 1H NMRspectroscopy that Hsp25 and a-crystallin have short, flexibleand solvent-exposed C-terminal extensions which protrudefrom the domain core of the molecule [9,10]. In mouse Hsp25,the C-terminal extension encompasses the last 18 amino acidswhich commences at an identical position, upon sequencealignment, to the flexible region observed in a-crystallin [10].The extensions share no sequence similarity but have thecommon characteristic of being polar. They also have noordered structure and conformational flexibility similar to thatof peptides of equivalent length [9±11], i.e. the extensions

Correspondence to J. A. Carver, Department of Chemistry,

University of Wollongong, Northfields Avenue, Wollongong, NSW 2522,

Australia. Fax: 1 61 24221 4287, Tel.: 1 61 24221 3340,

E-mail: [email protected]

Abbreviations: sHsp, small heat shock protein; Hsp25DC18, mutant of

mouse Hsp25 from which the C-terminal 18 amino acids are absent; ANS,

8-anilino-1-naphthalene sulphonate.

Enzyme: citrate synthase (EC 4.1.3.7).

Note: web site available at

http://www.uow.edu.au/science/chem/rijcarver.html

(Received 17 January 2000, accepted 28 January 2000)

1924 R. A. Lindner et al. (Eur. J. Biochem. 267) q FEBS 2000

adopt a statistically disordered conformation (sometimes calleda random coil), which has no defined secondary structure.

In a-crystallin, the primary role of the flexible C-terminalextensions is to act as solubilizers of the relatively hydrophobicprotein and the sHsp±substrate complex which forms uponchaperone action. Thus, a mutant of aA-crystallin in whichhydrophobicity was introduced into the C-terminal extensionhad significantly reduced flexibility within this extension,and a corresponding reduction in solubility, heat stability, andchaperone ability, compared with the wild-type protein [12].Furthermore in a-crystallin, upon formation of the sHsp±substrate complex during chaperone action, the flexibility of theC-terminal extension of aA-crystallin is maintained whilst thatof the extension of aB-crystallin is at least partially lost[13±15]. Thus, the latter extension may have some preferentialrole in interacting with the substrate protein.

In this paper, the role of the C-terminal extension in thestructure and chaperone action of mouse Hsp25 is probedthrough the preparation of a mutant of mouse Hsp25 in whichthe last 18 amino acids of the C-terminal extension are absent(Hsp25DC18). The structural and functional alterations under-gone by Hsp25DC18 are then investigated by a variety ofspectroscopic techniques.

M A T E R I A L S A N D M E T H O D S

Materials

Bovine a-lactalbumin (Ca21-depleted) (L-6010), dithiothreitoland 8-anilino-1-naphthalene sulfonate (ANS) were obtainedfrom Sigma. D2O (99.9% deuterated) and 98% deuteratedd10-dithiothreitol were obtained from Cambridge IsotopeLaboratories. Bovine a-crystallin was purified as describedby Slingsby and Bateman [16]. Recombinant mouse Hsp25 wasexpressed and purified as described by Engel et al. [17]. Citratesynthase from pig heart (EC 4.1.3.7) was obtained fromBoehringer Mannheim.

Site-directed mutagenesis and expression of Hsp25DC18

The mutant of Hsp25 lacking the C-terminal extension,Hsp25DC18, was constructed by oligonucleotide-directedmutagenesis using the Transformer site-directed mutagenesiskit (Stratagene), the template vector pAK3038Hsp25 and theC-terminal mutagenic primer 5 0-CCCCAATTTGGGCCTAG-GCCTCGAAAGTAAC-3 0. The correct mutated sequence wasverified by DNA sequencing. Expression and purification wasaccording to Buchner et al. [18]. In SDS/PAGE the truncatedprotein showed an apparent molecular mass of 20±21 kDa.This is as expected as the subunit mass of wild-type Hsp25 is23 014 Da and removal of 18 C-terminal amino acids results ina mass loss of < 1700 Da.

Citrate synthase aggregation assay

Citrate synthase (15 mm) diluted 1 : 200 in 40 mm Hepes/KOH,pH 7.5, was equilibrated at 43 8C in the presence and absenceof Hsp25 and Hsp25DC18. IgG (MAK33) was used as a controlfor unspecific protein effects. To monitor the kinetics ofthermal aggregation, light scattering was measured in a PerkinElmer MPF44A luminescence spectrophotometer in stirred andthermostatically controlled quartz cells. During the measure-ments both the excitation and emission wavelength were set to500 nm with a spectral bandwidth of 2 nm. Concentrations of

citrate synthase refer to the dimer, those of Hsp25 refer to themonomer.

a-Lactalbumin aggregation assay

The chaperone interaction of Hsp25 and Hsp25DC18 withdithiothreitol-reduced a-lactalbumin was monitored by visibleabsorption spectroscopy, based on the method of Farahbakhshet al. [19]. a-Lactalbumin (2 mg´mL21 in 20 or 50 mm sodiumphosphate pH 7.0, 0.1 m NaCl, 0.02% NaN3) was incubatedat 37 8C or 43 8C in the presence of a range of concen-trations of Hsp25, Hsp25DC18 or a-crystallin as a control(0±4 mg´mL21). The increase in light scattering at 360 nmfollowing addition of dithiothreitol (to a final concentration of20 mm) was monitored with time at 37 8C or 43 8C using aMolecular Devices Spectramax 250 spectrophotometer.

CD spectroscopy

CD studies were performed at room temperature on a JascoJ-720 spectropolarimeter calibrated with a-d10-camphor-sulfonic acid. Mean residue ellipticities (u) were calculatedusing a mean residue mass of 110 Da. Spectra were measuredwith a cell of pathlength 0.1 cm and 0.5 cm for far and nearUV, respectively. The secondary structure content was calcu-lated with the variable selection method [20] using the programVARSLC1 and taking into account a set of 33 referenceproteins.

Ultracentrifugation

Molecular mass determinations were carried out using an X-LAanalytical ultracentrifuge (Beckman Instruments) equippedwith absorbance optics. The molecular mass data were derivedeither from sedimentation equilibrium experiments at 10 8Cusing the program polymole [21] or by sedimentation velocityruns at 20 8C. The latter methodical variant allows simul-taneous calculation of sedimentation and diffusion coefficientsby fitting the time-dependent recorded radial concentrationprofiles using the program lamm [22]. From sedimentation anddiffusion coefficients, the molecular mass (Msd) was calculated.The frictional ratio, f / f0, and the diameter of the monomer wereestimated as discussed earlier [6]. All experiments wererepeated 7±10 times. The data presented are averages ofthese runs.

Electron microscopy

Negative staining was carried out with 1% uranyl formiateusing a double-layer carbon technique as described previously[6]. Micrographs were taken with an EM400T electron micro-scope (Philips Electron Optics, Eindhoven, the Netherlands) at80 kV and a magnification of 60 000�.

SDS/PAGE

SDS/PAGE was performed using the discontinuous buffersystem as described by Laemmli [23]. Protein bands werestained with Coomassie blue R250.

1H NMR spectroscopy1H NMR spectroscopy was undertaken on Hsp25DC18 (1.6 mmon a subunit basis) in 20 mm sodium phosphate, 0.02% NaN3,pH 6.0 and 90% H2O/10% D2O. Two-dimensional 1H NMR

q FEBS 2000 C-terminal flexibility and chaperone function of Hsp25 (Eur. J. Biochem. 267) 1925

experiments of Hsp25DC18 were performed at 600 MHz on aBruker DMX-600 spectrometer at 25 8C using the parametersoutlined in Werten et al. [24]. Spectra were acquired with 512 t1increments over 2048 t2 points, with 64 scans for TOCSYexperiments (with mixing times of 32 and 65 ms) and 80 scansfor the NOESY experiment (mixing time of 120 ms). Spectrawere processed using Felix 95 software (MSI). Chemical shiftswere referenced relative to H2O at 4.81 p.p.m.

Two-dimensional 1H NMR spectroscopy experiments(TOCSY) of wild-type Hsp25 alone and with a-lactalbuminin the presence and absence of 20 mm deuterated dithiothreitolwere performed at 400 MHz using a Varian Unity-400spectrometer as described previously [3,9].

ANS fluorescence binding experiments

The fluorescence of the hydrophobic probe, ANS, in thepresence of Hsp25DC18, Hsp25 and a-crystallin was monitoredat 37 8C using a Hitachi F-4500 fluorescence spectrophoto-meter with a circulating water bath. An excitation wavelengthof 387 nm was used and the emission spectrum was recordedfrom 420 to 540 nm. The maximum fluorescence occurred at479 nm for all samples. Samples contained 0.2 mg´mL21 ofprotein and ANS to a final concentration of 15 mm.

Visible absorption monitoring of protein heat stability

Samples (Hsp25DC18, Hsp25 and a-crystallin) at a concen-tration of 0.4 mg´mL21 were placed in a 1-cm pathlength,thermostatically controlled quartz cell. Light scattering at360 nm was measured with increasing temperature from25 8C to 66 8C in a Shimadzu UV-2401PC spectrophotometer.

R E S U LT S

Comparison of structural characteristics of wild-type Hsp25and Hsp25DC18 using CD, analytical ultracentrifugation andelectron microscopy

The CD spectrum of Hsp25DC18 in the far-UV region wassimilar to that of the wild-type protein [25], i.e. both proteinsgave a major minimum at around 218 nm, consistent withmainly b-pleated sheet character (Fig. 1). Compared to Hsp25,however, Hsp25DC18 also had a relatively strong shoulderat 208 nm in the region expected for a-helical secondarystructure. Indeed, deconvolution of both spectra gave secondarystructure contents of 40% b-pleated sheet and 11% a-helix forHsp25DC18 compared to 46% b-pleated sheet and 2% a-helixfor wild-type Hsp25. CD spectra acquired in the near UVregion were qualitatively identical for the wild-type and mutantprotein (data not shown). A decrease in intensity of signal(< 35% at 280 nm) was observed for Hsp25DC18 indicating

that some reduction in ordered conformation around aromaticside chains and thus tertiary structure occurs as a result ofC-terminal cleavage.

The molecular mass of Hsp25DC18 determined from bothsedimentation equilibrium and sedimentation velocity experi-ments is approximately 650 kDa (Table 1). Considering thatthe removal of 18 C-terminal amino acids decreases the mol-ecular mass of the monomers (M1) by about 1700 Da, theassociated state of 30±32 monomers in the Hsp25 complex isconserved in Hsp25DC18 (M/M1 in Table 1). The sedimenta-tion and diffusion coefficients of Hsp25DC18 and Hsp25 aresimilar (Table 1). As a result, the frictional ratio, f / f0, ofHsp25DC18 deviates only marginally from the wild-type value(Table 1) which suggests that nearly globular complexes ofHsp25DC18 of 15±16 nm in diameter are present [6]. In thiscalculation, it is assumed that the absence of the C-terminal 18amino acids in Hsp25DC18 does not lead to a significantchange in hydration compared to Hsp25. In agreement with theultracentrifugation data, electron micrographs of Hsp25DC18showed that the mutant formed very similar oligomericstructures to those formed by the wild-type protein (Fig. 2),i.e. roughly spherical particles of < 15 nm in diameter wereobserved indicating that the C-terminal extension of 18 aminoacids plays no major role in formation of multimeric Hsp25particles. Thus, in summary, removal of the C-terminal exten-sion of Hsp25 has some effect on the secondary and tertiarystructure of the protein but does not affect its oligomeric state.

Fig. 1. Far UV CD spectra of Hsp25DC18 and Hsp25. CD spectra for

Hsp25DC18 (solid line, 0.25 mg´mL21) and Hsp25 (broken line,

0.16 mg´mL21) were recorded in 10 mm potassium phosphate buffer,

pH 7.4, at 20 8C. For Hsp25DC18, the spectra show features characteristic

for a mainly b-pleated sheet protein (11 ^ 1% a-helix, 40 ^ 1% b-pleated

sheet, 18 ^ 1% turn). Similarly, analysis for wild-type Hsp25 revealed

< 2% a-helix, 46% b-pleated sheet, 18% turn.

Table 1. Hydrodynamic data for Hsp25DC18 and Hsp25. MsD, Molecular mass calculated from sedimentation (s20,w) and diffusion (D20,w) coefficients;

Mw, weight average molecular mass derived from sedimentation equilibrium runs; M, for Hsp25DC18, the average of MsD and Mw (i.e. 649 kDa): for Hsp25,

M � MsD, as Mw was not determined [6]; M1, monomeric molecular mass; nd, not determined.

Protein s20,w (S)

D20,w

(107 cm2´s21)

Msd

(kDa)

Mw

(kDa) f / f0 M/M1 Reference

Hsp25C18 20�.7 ^ 0.3 2�.97 ^ 0.07 645 ^ 24 653 ^ 25 1�.25 30�.3 This study

Hsp25 21�.2 ^ 0.3 2�.64 ^ 0.08 730 ^ 25 ND 1�.35 31�.8 6


Comparison of the chaperone activity of wild-type Hsp25and Hsp25DC18

The chaperone ability of Hsp25 following removal of itsC-terminal extension was compared to that of the wild-typeprotein using two different assays, one involving thermalaggregation and subsequent precipitation of citrate synthaseat 43 8C [4] and the other monitoring the precipitation ofa-lactalbumin following reduction of its four disulfide bondswith dithiothreitol [26,27]. In both assays, the light scatteringfrom the precipitating particles was monitored spectrophoto-metrically. The ability of Hsp25DC18 to prevent the heat-induced precipitation of citrate synthase was found to becomparable to that of the wild-type protein (Fig. 3). In thesecond assay, the ability of Hsp25DC18 to prevent thedithiothreitol-induced precipitation of a-lactalbumin at 37 8Cwas compared to wild-type Hsp25 and a-crystallin. In thepresence of increasing amounts of a-crystallin [27] and Hsp25(Fig. 4A), this precipitation can be reduced and, at sufficientlyhigh concentrations of these sHsps, prevented. As is shown inFig. 4B, however, Hsp25DC18 did not stabilize a-lactalbuminin such an assay. In fact, with increasing amounts ofHsp25DC18, a proportional increase in precipitation of proteinwas observed suggesting that in addition to the precipitation ofa-lactalbumin, Hsp25DC18 also precipitated out of solutionupon addition of dithiothreitol. As a control, Hsp25DC18 in theabsence of a-lactalbumin was stable following addition ofdithiothreitol. Likewise, a-crystallin was not affected by theaddition of dithiothreitol and readily prevented a-lactalbuminfrom precipitation following addition of dithiothreitol (Fig. 4).The stability and functional properties of wild-type Hsp25are also unaffected by the addition of dithiothreitol [28].SDS/PAGE of the supernatant and pellet of the sample ofHsp25DC18 in the presence of a-lactalbumin and dithiothreitolconfirmed that Hsp25DC18 precipitated under these condi-tions, i.e. Hsp25DC18 was present in the pellet along witha-lactalbumin with virtually no Hsp25DC18 in the supernatant(data not shown). The lack of precipitation of Hsp25DC18 uponthe addition of dithiothreitol in the absence of substrate, but itsprecipitation under reducing conditions in the presence ofsubstrate, suggests that in the latter case, Hsp25DC18 isprecipitating as part of a sHsp±substrate complex. SDS/PAGEof a control sample containing a-crystallin and a-lactalbumin(1 : 1, w/w) in the presence of dithiothreitol did not contain anya-crystallin in the pellet (data not shown).

The chaperone ability of a-crystallin increases at highertemperatures [29,30]. The dithiothreitol-induced aggregationassay for a-lactalbumin was therefore repeated at 43 8C todetermine if the different chaperone abilities of Hsp25DC18

under reducing and heating conditions could be explained bythe different temperatures used for each assay. Very similarresults were obtained at 43 8C to those at 37 8C and are alsopresented in Fig. 4B, i.e. no protection of a-lactalbumin fromdithiothreitol-induced precipitation was observed in the pre-sence of Hsp25DC18, which was accompanied by precipitationof both proteins. Thus, temperature was not a factor indetermining the different chaperone ability of Hsp25DC18 inthe two assays.

Hsp25DC18 shows reduced temperature stability andexposed hydrophobicity compared with wild-type Hsp25

Fluorescence experiments using the hydrophobic probe ANSshowed that Hsp25DC18 has less clustered hydrophobicityexposed to solution compared with wild-type Hsp25 anda-crystallin, with Hsp25DC18 (85.8 ^ 0.5 units) exhibitingmuch less ANS fluorescence than a-crystallin (165.2 ^ 0.9units) and wild-type Hsp25 at 37 8C (125.2 ^ 1.2 units) at the

Fig. 3. Suppression of thermal aggregation of citrate synthase in the

presence of Hsp25 and Hsp25DC18 at 43 8C. Citrate synthase (final

concentration: 75 nm; dimer) was diluted into thermostatically controlled

solutions containing 18 nm Hsp25 (K), 18 nm Hsp25DC18 (O), 75 nm

Hsp25 (S), 75 nm Hsp25DC18 (V) or 1.3 mm IgG (A). Open circles (W)

represent the spontaneous aggregation of citrate synthase at 43 8C. The

kinetics of aggregation were determined by measuring the light scattering

of the samples at 500 nm.

Fig. 2. Electron micrographs of (A) negatively

stained wild-type Hsp25 and (B) Hsp25DC18

complexes. Original magnification 60 000�.


same concentration (as determined using the Bradford proteinassay [31]) and 37 8C. In addition, Hsp25DC18 was much lesstemperature stable compared with the other two proteins. Bymonitoring the light scattering at 360 nm with increasingtemperature of a 0.2 mg´mL21 solution of Hsp25DC18, theprotein was observed to aggregate and precipitate irreversiblyout of solution at around 55 8C (data not shown). The mutantof aA-crystallin containing additional hydrophobicity in itsC-terminal extension behaved very similarly in aggregating andprecipitating at around the same temperature [12]. In contrast,wild-type Hsp25 and a-crystallin were stable up to much highertemperatures (to 65 8C) [12,29,30].

1H NMR spectroscopy of Hsp25DC18 reveals new regions ofconformational flexibility

To gain detailed information about the nature of the structuralchanges undergone in Hsp25 upon deletion of its C-terminalextension, 1H NMR spectroscopic studies were undertaken onHsp25DC18. The one- and two-dimensional 1H NMR spectraof Hsp25DC18 revealed a significant number of resonanceswhich reflects some additional flexibility in the moleculefollowing removal of its C-terminal extension. In Hsp25, theseresonances were not observed [10]. Cross-peaks in the TOCSYspectrum originating from the NH protons of Hsp25DC18 are

Fig. 4. The chaperone activity of Hsp25DC18, a-crystallin and Hsp25 as measured by their inhibition of dithiothreitol-induced precipitation of

a-lactalbumin. Precipitation was monitored by the increase in absorbance at 360 nm. (A) A comparison of the chaperone activity of a-crystallin and Hsp25.

All samples were in 20 mm phosphate buffer, pH 6.9, 0.1 m NaCl, 0.02% NaN3, at 37 8C. Precipitation of a-lactalbumin was induced by the addition of

dithiothreitol to a final concentration of 20 mm. (B) A comparison of the chaperone activity of Hsp25DC18 and a-crystallin. All samples were in 50 mm

phosphate pH 6.9, 0.1 m NaCl, 0.02% NaN3, at 37 8C (with the exception of data denoted by K, which were acquired at 43 8C). Precipitation was induced by

the addition of dithiothreitol to a final concentration of 20 mm. The control solutions of a-crystallin and Hsp25DC18 were at 2 mg´mL21. DTT, dithiothreitol.


shown in Fig. 5. The absence of chemical shift dispersion of thea-CH resonances and hence the resultant overlap in thespectrum implied that the cross-peaks arose from regions inthe molecule that were flexible and had little ordered structure.

Despite the extensive overlap in the spectrum it was possibleto link strong cross-peaks arising from a Glu±Ala dipeptidetogether via standard sequential assignment methods [32] usinga combination of the TOCSY and NOESY spectra. There aretwo Glu±Ala dipeptide sequences in mouse Hsp25DC18 (atresidues 166 167 and 186 187) and therefore either of theseamino acid pairs could give rise to the well-resolved cross-peaks labelled in Fig. 5. Residue 168 is a proline which, in anunstructured region, should give a major downfield shift to thea-CH chemical shift of Ala167 to around 4.62 p.p.m. [33]. Infact, the a-CH chemical shift of the alanine cross-peak in Fig. 5is 4.36 p.p.m. which is that expected for an alanine residue in arandom coil conformation when not followed by a proline [33].Thus, cross-peaks due to the Glu±Ala dipeptide are assignedtentatively to the two C-terminal residues of Hsp25DC18(Glu186 and Ala187). In addition to these cross-peaks, intra-residue cross-peaks are also observed for many other aminoacids, e.g. cross-peaks for six alanines, three glutamic

acids/glutamines, two valines, two aromatic residues and athreonine are indicated in Fig. 5. Assigning these cross-peaks tospecific residues was not possible due to extensive overlap inthe NOESY spectrum. Although the putative C-terminaldomain of mouse Hsp25DC18 from Glu87 to Ala187, asaligned by Caspers et al. [34], has six alanine amino acidswhilst the putative N-terminal domain contains 14 alanineamino acids, it is unlikely that the cross-peaks observed in the1H NMR spectra of Hsp25DC18 arise from the entireC-terminal domain of the protein, i.e. 53% of the monomer,since the overall hydrodynamic properties and quaternarystructure of the mutant are similar to those of the wild-typeprotein (Table 1, Fig. 2). More likely, the cross-peaks observedin Fig. 5 arise from flexible residues in Hsp25DC18 located inor near the region or regions of C-terminal flexibility whichexhibit chemical shift inequivalence due to local conforma-tional variation. This conformational variability could berelated to a looser organization of the exposed hydrophobicsurface of the mutant protein. The added flexibility observed byNMR spectrosopy may also account for the reduction inordered tertiary structure observed in the near UV region of theCD spectrum of Hsp25DC18. Indeed a number of aromatic

Fig. 5. Cross-peaks arising from the NH

protons in a 1H NMR TOCSY spectrum

(mixing time� 65 ms) of Hsp25DC18 acquired

at 600 MHz. The assignments for cross-peaks

from Glu186 and Ala187 are tentative (see text).

Data were acquired at 25 8C and the sample was

in 20 mm sodium phosphate buffer, pH 6.0,

0.02% NaN3, and 90%H2O/10%D2O.


residues (phenylalanine, tryptophan) were observed by two-dimensional NMR (Fig. 5) indicating a reduction in conforma-tional order around these residues. Thus, in summary, comparedwith Hsp25, Hsp25DC18 exhibits extra flexibility in thevicinity of its `new' C-terminus which is not present in thewild-type protein.

The C-terminal extension of Hsp25: does it interact directlywith the bound substrate during chaperone activity?

To investigate the role of the flexible C-terminal extension inchaperone action, the interaction of bovine a-lactalbumin withmouse Hsp25 was monitored by 1H NMR spectroscopy. Asdetermined by size exclusion HPLC, the stabilization ofreduced a-lactalbumin by a-crystallin during its chaperoneaction arises from the formation of a sHsp±substrate complexbetween the two proteins [15,27]. Figure 6A shows thealiphatic region of the 1H NMR TOCSY spectrum of Hsp25.The cross-peaks have been assigned previously [10] and

correspond to the 18-amino-acid C-terminal extension ofHsp25. Spectra were also acquired on a 1 : 2 (w/w) mixtureof a-lactalbumin/Hsp25 in which, in addition to the cross-peaksarising from Hsp25, a multitude of cross-peaks were alsoobserved from monomeric a-lactalbumin (mass < 14 kDa). Tothis sample, 20 mm dithiothreitol was added and the one-dimensional 1H NMR spectrum was monitored as the Hsp25-a-lactalbumin complex formed [15,27]. Two hours after additionof dithiothreitol (by which time the reaction was deemed tohave reached completion as judged by no further change in theone-dimensional spectrum), a TOCSY experiment was per-formed on the sample (Fig. 6B). Only two broad cross-peakswere observed from a-lactalbumin due to the immobilization ofthe substrate as a result of its incorporation into the sHsp±substrate complex. However, no change in the number of cross-peaks arising from the C-terminal extension of Hsp25 occurredupon formation of the soluble complex with a-lactalbuminupon addition of dithiothreitol (cf. Fig. 6A,B). There is a slightbroadening of peaks in Fig. 6B which may be a result of

Fig. 6. Two-dimensional 1H NMR TOCSY

spectrum (mixing time, 30 ms) showing

cross-peaks arising from the a-CH protons

of (A) Hsp25 and (B) a 1 : 2 (w/w) mixture of

a-lactalbumin:Hsp25 following addition of

dithiothreitol to a final concentration of

20 mmm. Data were acquired at 400 MHz and

37 8C in 20 mm sodium phosphate pH 7.0,

0.1 m NaCl, 0.02% NaN3, D2O. Cross-peaks

previously assigned to the C-terminal extension

of Hsp25 [10] are labelled in (B).


formation of the high molecular mass complex between the twoproteins. This complex will have an overall slower tumblingrate compared with that of Hsp25 alone. Under the conditionsof this experiment, Hsp25 will be saturated with substrate assome precipitation of a-lactalbumin is still observed by visibleabsorption spectroscopy at a 1 : 2 (w/w) ratio of a-lactalbumin/Hsp25 (Fig. 4A). Indeed, a precipitate was observed in theNMR tube following the completion of the experiment. It isconcluded that upon interaction of Hsp25 with a-lactalbuminduring the chaperone action to form the sHsp±substratecomplex, the flexibility of the C-terminal extension of Hsp25is maintained, i.e. the C-terminal extension of Hsp25 is notinvolved in direct association with a-lactalbumin.

D I S C U S S I O N

A polar, highly flexible C-terminal extension appears to be aconserved feature of mammalian sHsps [34]. In this investiga-tion, this extension was removed in Hsp25 in order to ascertainits role in the structure and function of sHsps. The resultingmutant showed little quaternary structural alteration, formingsphere-like aggregates comprising 30±32 monomers (i.e.comparable to the wild-type protein), but there was somealteration in the secondary and tertiary structure of the protein(e.g. a slight reduction in b-pleated sheet) and in the overallstructural integrity as shown by the reduction in exposedhydrophobicity of the mutant.

The flexibility of the C-terminal extension of wild-typeHsp25 was maintained upon complex formation with thedestabilized substrate protein, a-lactalbumin, indicating thatit does not interact with the bound substrate protein. Ina-crystallin, sHsp±substrate complex formation leads to themaintenance of flexibility in at least one of the C-terminalextensions. Thus, the flexibility of the aA-crystallin extensionis preferentially maintained, over that of the aB-crystallinextension, in formation of the sHsp±substrate complex withdestabilized, heated g-crystallin [14] and in the naturallyoccurring sHsp±substrate complex that is found in older lenses[13]. However, the flexibility of extensions of both subunits ina-crystallin is retained upon the formation of the sHsp±substrate complex under reducing conditions [15]. From theseresults, it can be concluded that C-terminal flexibility is animportant feature for the chaperone activity of mammaliansHsps.

In agreement with this, studies with a mutant ofaA-crystallin in which hydrophobicity was introduced into theC-terminal extension resulted in a loss of flexibility and aconcomitant loss in solubility and chaperone activity [12].The need for C-terminal flexibility in Hsp25 is also apparentfrom the observation that removal of the extension in theHsp25DC18 mutant leads to the introduction of additionalflexibility in the C-terminal region which does not causesignificant alteration in the quaternary structure of the protein,but maintains at least partial chaperone activity. We proposethat the principal role of these extensions in sHsps is to act aspolar solubilizing agents for the relatively hydrophobic sHspproteins and the sHsp±substrate complex which forms uponchaperone action; they are not involved in binding directly tothe substrate. They may also serve as `spacers' in preventingsHsp±substrate complexes from approaching one anotherwhich, without their presence, would result in aggregationand precipitation due to hydrophobic interactions between theproteins. Because of the flexibility of these extensions, theirsolubilizing function conceivably arises from an entropiccontribution to the free energy of the solution state. If the

polarity and flexibility of the extensions are disrupted, as in thehydrophobic mutant of aA-crystallin [12], the stability andchaperone activity of the protein are reduced significantly.

Induction of extra C-terminal flexibility in Hsp25DC18 doesnot compensate entirely for the loss of the natural C-terminalextension of Hsp25, as judged from the decreased thermalstability and partially impaired chaperone ability of themutant. Thus, Hsp25DC18 is ineffective as a chaperone witha-lactalbumin under reducing conditions at 37 8C and 43 8Cbut has comparable chaperone activity to the wild-type proteinin a heat denaturation assay with citrate synthase at 43 8C(Fig. 3). Other researchers have observed differences in chape-rone activity of aA-and aB-crystallin when comparing theheat-protection and dithiothreitol-induced reduction assays[35], i.e. both phosphorylation and carbamylation were foundto enhance chaperone activity as measured by dithiothreitol-induced reduction, whilst the heat-protection capabilitiesremained unaffected. Likewise, the chaperone ability of theindividual aA-and aB-crystallin aggregates is different in theheating and reduction assays [36], e.g. at physiologicaltemperature, aB-crystallin is a better chaperone in thereduction assay than aA-crystallin, whilst in the heatingassay aA-crystallin displays better chaperone activity thanaB-crystallin. This difference in activity is attributed to thegreater thermal stability of aA-crystallin compared to that ofaB-crystallin. Clearly the nature of the chaperone ability ofHsp25DC18 is challenged differently under these two denatur-ing conditions. We observe that under reducing conditions, thenewly induced C-terminal flexibility in Hsp25DC18 isinsufficient to solubilize the sHsp±substrate complexes thatform as a result of addition of dithiothreitol. Consequently,these complexes aggregate and precipitate from solution.Compared to heat-denatured citrate synthase, dithiothreitol-reduced a-lactalbumin may expose more hydrophobicitymaking it more susceptible to aggregation and precipitationwhen bound to Hsp25DC18. For a chaperone to be effective,chaperone±solvent interactions that are sufficient to stabilizeand solubilize the isolated chaperone oligomer must besufficient also for solubilizing the chaperone±substrate com-plex (in the absence of gross conformational changes in theformer). Unfortunately, it was not possible to compare thechaperone ability of Hsp25DC18 under the two assay condi-tions of heat and reduction with the same substrate protein (e.g.ovotransferrin which precipitates both upon heating andreduction [15]) because Hsp25DC18 precipitates out of solutionat around 55 8C, well below the temperature at whichovotransferrin precipitates.

That Hsp25DC18 exhibits lower thermal stability comparedwith Hsp25, in spite of having a similar oligomeric state to thatof the wild-type protein, is most probably related to thereduction in clustered surface hydrophobicity (inferred fromANS fluorescence measurements). Reduction in the clusteredhydrophobic surface means breaking the continuity of theexposed hydrophobic patches that may be involved in actingeither against thermal denaturation or in favour of hydrophobicbinding of denatured substrate proteins [37]. In addition,reducing the hydrophobic surface clustering alters the orienta-tion of the water molecules adjacent to the hydrophobic surfacewhich increases the degree of solvent ordering [37]. As a result,the entropy of the system is reduced, an effect that inHsp25DC18 is only partially balanced by introducing extramobility in the C-terminal region. Thus, it appears thatremoving the flexible C-terminal extension of Hsp25 causes amodification of the hydrophobic surface organization of themolecule which undermines its chaperone performance,


especially when the maximal hydrophobic binding capability isneeded to cope successfully with binding of dithiothreitol-reduced a-lactalbumin.

Leroux et al. [38] have prepared a mutant of a Caenorhab-ditis elegans sHsp, Hsp16-2, with its last 16 amino acidsremoved. In concordance with our results, this mutant formssimilarly sized oligomeric complexes and also has identicalchaperone action to that of wild-type Hsp16-2 in the citratesynthase heat assay. Compared with the wild-type protein,however, the mutant was much less stable to a freeze±thawingcycle. These data are consistent with the notion that theC-terminal extension in sHsps is not directly involved in thechaperone action but acts as a stabilizing and solubilizing agentfor the protein and the sHsp±substrate complex.

Arrangement of the domains in sHsps as predicted byWistow [39] suggests that the flexible C-terminal region inmouse Hsp25 should extend from Thr174 to Lys205 [34], i.e.over a wider region than that observed experimentally (Arg188to Lys205 [10]). The extra flexibility observed for Hsp25DC18(Fig. 5) is consistent with this postulate. In fact, as predicted byWistow [39], the potentially flexible C-terminal region ofHsp25 contains a section of b-pleated sheet from Ile179 toAla187, i.e. just before the start of the C-terminal extension(Arg188 to Lys205). Indeed, the crystal structure ofM. jannaschii Hsp16.5 shows that the comparable region at itsC-terminus adopts an extended (b-pleated sheet) structurewhich reaches out and interacts with a neighbouring subunit[7]. Thus, in Hsp25, this b-pleated sheet should be disruptedwith removal of the C-terminal extension leading to an increasein flexibility of the C-terminal region. The loss of this region ofb-pleated sheet therefore could account, at least in part, for thereduction of b-pleated sheet secondary structure as observed byCD spectroscopy (Fig. 1). That this region, albeit relativelyrigid in Hsp25, is located in a solvent-accessible boundaryregion is also consistent with the observation that inaA-crystallin, the Arg157±Ala158 bond, which upon align-ment corresponds to Ile179±Thr180 in Hsp25 [34], is highlysusceptible to cleavage by trypsin [40].

In summary, removal of the flexible C-terminal extension ofHsp25 was shown to have no effect on the quaternary structureof Hsp25. The modification, however, introduced alteration inits secondary and tertiary structure, additional C-terminalflexibility and a reduction in the clustered, exposed hydro-phobicity of the molecule. Chaperone activity was comparablebetween the wild-type and mutant for preventing heat denatu-ration of citrate synthase at 43 8C. In contrast, the mutant wasfound to be less stable at higher temperatures compared withthe wild-type protein and was unable to prevent dithiothreitol-induced precipitation of a-lactalbumin. Thus, it is concludedthat a flexible C-terminal extension is essential for completechaperone activity and stability of mammalian sHsps.

A C K N O W L E D G E M E N T S

This work was supported by grants from the National Health and Medical

Research Council of Australia to J. A. C. and from the German Ministry of

Education and Research to M. G. and J. Buchner.

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mouse hsp25, a small heat shock protein

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