Subunit Exchange of Multimeric Protein Complexes

Subunit Exchange of Multimeric Protein Complexes 38923FIG. 2.NanoESI mass spectra of thesmall plant heat shock proteinsTaHSP16.9 (A) and PsHSP18.1 (B) underconditions where noncovalent interactionsare preserved in the massspectrometer. Left panel, mass-tocharge(m/z) scale. Right panel, transformedto mass scale. The peaks representmultiply protonated dodecameric complexes.For the molecular masses of speciesa–d, see “Results.”individual aliquots were analyzed at different time intervals and theresulting spectra were found to be indistinguishable from those obtainedduring the continuous sampling experiment (data not shown).Kinetic Data—To extract kinetic information from the time-resolveddata, we combined spectra over 8-s intervals around 29 time points inaddition to the end point. Because of the overlap of a number of peaksin the mass spectrum that arises from the heterogeneity of the mixedoligomers, only the intensities (peak heights) of those signals, whichcould be assigned unambiguously, were taken into account. The followingpeaks were monitored: m/z 5906 and 5737 for (16.9) 12 ; m/z 6746 and6541 for (18.1) 12 ; m/z 6312 and 6130 for (18.1) 11 (16.9) 1 ; m/z 6669, 6466,and 6095 for (18.1) 10 (16.9) 2 ; m/z 6630, 6430, 6239, and 6059 for(18.1) 9 (16.9) 3 ; and m/z 6590, 6390, 6202, and 6024 for (18.1) 8 (16.9) 4 . Theintensities of each component were expressed as percentages of thetotal intensity, and the scale adjusted to the initial and final abundancesin solution (given by a theoretical binomial distribution) (seebelow). Initial decay rates were calculated by plotting the naturallogarithm of the intensity versus time and fitting linear lines of best fit.Simulation of Mass Spectra—The simulated spectrum was constructedusing SigmaPlot 2001 (SPSS Science, Chicago, IL). Theoreticalintensities of the 13 different dodecamers were calculated based on abinomial distribution of the components PsHSP18.1 and TaHSP16.9 ata ratio of 3:1, assuming an equal preference for each composition. Thestatistical propensity, P n , of individual dodecamers containing n timesthe 18.1 subunit is given by Equation 112!P n n!12 n! P 18.1 n P 16.9 12n (Eq. 1)where P 18.1 and P 16.9 are the relative abundances of HSP18.1 and 16.9subunits in solution (0.75 and 0.25, respectively, for a 3:1 ratio). Thelast two terms describe the probability of finding certain numbers ofeach subunit in the complex, whereas the first term accounts for thenumber of possible arrangements of these subunits within the complex.A three-parameter Gaussian curve was fitted across the tops of thepeaks in the spectra of the two individual sHSPs. The midpoint andwidth of these curves were used to create similar ones for the theoreticalcharge state distributions of the heterododecamers. Their maxima werescaled to the theoretical intensity of the various species. Three-parameterLorentzian curves were used to model individual peaks with a peakwidth at half-height of 10 Da as measured from the charge states in themass spectra of the homododecamers. The intensity was given by solutionof the Gaussian function for that charge state. Combining allsimulated peaks resulted in the total simulation.RESULTSCharacterization of sHSP Complexes—Fig. 2 shows nanoESImass spectra of the small heat shock proteins PsHSP18.1 andTaHSP16.9 under conditions optimized for the transmission ofintact noncovalent complexes. The dominant feature of bothspectra is the group of peaks around an m/z value of 6000 (leftpanels) with very little additional signal outside this range.Fig. 2, right panels, show a transformation of the peaks in thisrange from an m/z to a mass scale. For TaHSP16.9 (Fig. 2A),only one major species a is detected with a molecular mass of200,790 Da. This mass compares well with the mass calculatedfor 12 TaHSP16.9 monomers (200,663.6 Da). Therefore, weconclude that TaHSP16.9 forms a dodecamer in solution asexpected from its x-ray structure (Fig. 1) (18). The observationthat the experimental mass is slightly higher than that calculatedfrom the sequence (in this case by 126 Da) is common

38924Subunit Exchange of Multimeric Protein ComplexesFIG.3.Products of subunit exchangebetween PsHSP18.1 and TaHSP16.9 atmolar ratios of 6:1 (A) and 1:6 (B) afterequilibration of the mixture. The majorspecies are labeled for each charge state.The numbers in the colored circles indicatethe number of PsHSP18.1 subunits in theheterododecamers.for mass spectra of noncovalent complexes and is attributed tothe binding of solvent molecules and buffer ions, presumably atthe subunit interfaces (41).The mass spectrum recorded for a solution of PsHSP18.1 issimilar to that of TaHSP16.9, although the peaks appear at aslightly higher m/z and carry on average more charges (Fig.2B). Here we observe three distinct complexes of similar mass:215,850 (for b), 215,215 (for c), and 214,452 Da (for d). Again,the complexes consist of 12 subunits as seen by comparisonwith a theoretical mass for the dodecamer of 215,643.7 Da.More than one-third of the total PsHSP18.1 signal resides inpeaks c and d. The isolation of these species in the quadrupoleanalyzer of the instrument and subsequent collision-induceddissociation (CID) reveals that dodecamer b consists of 12identical copies of the full-length protein, whereas c and d eachcontain one subunit with a truncation of the first six (c) and 13(d) N-terminal amino acids, respectively (CID spectra notshown). These truncations are likely to have arisen duringprotein expression and purification. As their presence does notdestabilize the complex relative to CID (spectra not shown),they do not appear to compromise the formation of the dodecamerin these quantities.Products of Subunit Exchange—We used mass spectrometryto determine the products of the reaction between PsHSP18.1and TaHSP16.9 at four different molar ratios (6:1, 3:1, 1:3, and1:6). Spectra recorded after equilibration of mixtures at molarratios of 6:1 and 1:6 of the two sHSPs are shown in Fig. 3, A andB, respectively. Many peaks are observed that are not present

Subunit Exchange of Multimeric Protein Complexes 38925FIG. 4. Simulated and measuredelectrospray mass spectra of theproducts of subunit exchange betweenPsHSP18.1 and TaHSP16.9 at amolar ratio of 3:1. Theoretical intensitiesof the individual species were calculatedbased on a binomial distribution ofthe components as expected for freely interchangeablesubunits (see inset, cf. Eq.1). The summation over all species (lowerblack trace) is in close agreement with theexperimental spectrum (upper blacktrace).in the spectra of the individual sHSPs. Mass measurementshows that they arise from dodecamers containing subunits ofboth sHSPs. At a 6:1 ratio (Fig. 3A), peaks are observed thatcorrespond to a range of heterododecamers containing between8 and 12 PsHSP18.1 monomers, and the most intense signal isattributed to the species (18.1) 10 (16.9) 2 . The minor peaks notlabeled in this spectrum arise from heterododecamers containingtruncated PsHSP18.1 subunits. For the opposite 1:6 ratio,dodecamers can be seen that contain between 8 and 12TaHSP16.9 monomers (Fig. 3B). Here the species with thehighest intensity is (18.1) 2 (16.9) 10 . The charge state ensemblehas shifted to lower m/z values relative to the 6:1 ratio becauseof the presence of fewer PsHSP18.1 monomers within the dodecamers.The comparatively low PsHSP18.1 concentrationalso explains the apparent absence of peaks, which are becauseof dodecamers containing truncated subunits. Similarly, at18.1:16.9 ratios of 3:1 (Fig. 4A, upper part) and 1:3 (data notshown), we find the most intense species to be (18.1) 9 (16.9) 3and (18.1) 3 (16.9) 9 , respectively, after equilibration.For the mixtures studied above, it appears that the intensitiesof heterododecamers formed by subunit exchange is governedby the relative concentration of the individual sHSPs insolution. In other words, the distribution of heterododecamersappears to be centered on the ratio of the two components in themixture. For example, the most abundant heterododecamerformed from an 18.1:16.9 ratio of 3:1 is (18.1) 9 (16.9) 3 . To establishwhether the distribution of the different heterocomplexesis determined solely by the abundance of the sHSP subunits insolution, we simulated a “statistical” spectrum, assuming nopreference for the uptake of monomers of either sHSP nor anyinherent preference for particular stoichiometries. The histogramin Fig. 4, inset, shows the theoretical binomial distributionof heterocomplexes for an 18.1:16.9 ratio of 3:1 according toEquation 1. From this distribution, it can be seen that thedominant species is indeed (18.1) 9 (16.9) 3 among a broad rangeof different compositions ranging from (18.1) 4 (16.9) 8 (n 4) tothe homododecamer (18.1) 12 (n 12). Using the theoreticalabundances of the different species from the histogram andtheir theoretical charge state distribution (see “Experimentalprocedures”), we plotted simulated ESI spectra for all dodecamers(Fig. 4, lower part). The sum of all of these overlappingspecies corresponds well to the actual mass spectrum recordedfor a 3:1 ratio (Fig. 4, upper part). The major difference betweenthe simulated and experimental data is the appearance of ahigher background signal, which we attribute primarily tooverlapping peaks arising from multiple charge states of bothfull-length and truncated PsHSP18.1 subunits with coincidentm/z values that were not taken into account in the simulation.The fact that the distribution of species matches that predictedby the simulation supports the picture of a statistical incorporationof different subunits into the dodecamer.Kinetics of Subunit Exchange—We followed the kinetics ofthe subunit exchange between HSP18.1 and 16.9 by monitoringthe reaction continuously over a period of 40 min to elucidatedetails of the reaction mechanism. Fig. 5 shows three timepoints recorded for a solution of 18.1:16.9 at a ratio of 3:1. Inthis experiment, the spectra have been averaged over 4-minintervals to improve signal-to-noise ratios. The t 0 spectrumrepresents a superposition of HSP18.1 and 16.9 spectra (cf. Fig.2, A and B) with their relative intensities adjusted according totheir molar ratio. The first indication of subunit exchange isapparent after 2 min when the signal of the heterocomplex(18.1) 10 (16.9) 2 is clearly observed (spectra not shown), and after8–12 min, this species dominates the spectrum. Eventually,after 36–40 min, we observe the equilibrium distribution ofheterocomplexes as described above.The intensities of the peaks assigned to (18.1) 12 and (16.9) 12were plotted as a function of time in Fig. 6. The abundance ofthese homododecamers follows an exponential decay functionover approximately the first 10 min, consistent with a firstorderreaction. By plotting the natural logarithm of this decayat the beginning of the reaction (Fig. 6, inset), we can extractthe rate constants for dissociation from the slope of the linearline of best fit. They are 0.24 0.05 and 0.20 0.04 min 1 at24 °C for HSP18.1 and 16.9, respectively. After 10 min, veryfew of the homododecamers remain, and a variety of heterocomplexeshas been formed. The relative abundances of thefour predominant heterospecies are plotted in Fig. 7. From thedata, it can be seen that heterocomplexes containing an evennumber of each type of subunits are formed more rapidly than

38926Subunit Exchange of Multimeric Protein ComplexesFIG. 5. Subunit exchange betweenPsHSP18.1 and TaHSP16.9 at a molarratio of 3:1 monitored in real-time bymass spectrometry. The labels are thesame as indicated for Fig. 3. For t 0(A)spectra of HSP18.1 and 16.9 have beensuperimposed using MassLynx with theintensities adjusted for their molar ratios.B and C, the dodecamer distribution averagedover a 4-min interval around 10and 38 min, respectively.those containing an odd number, i.e. (18.1) 10 (16.9) 2 is producedfaster than (18.1) 11 (16.9) 1 , and (18.1) 8 (16.9) 4 is produced fasterthan (18.1) 9 (16.9) 3 . All of the curves with the exception of(18.1) 9 (16.9) 3 go through a maximum before they reach theirequilibrium levels. Whereas a simple look at the abundance ofeach species at equilibrium (see above) suggests a purely statisticalexchange of subunits, the evolution of specific heterocomplexesreveals a different picture, that of even-numberedspecies being kinetically favored.DISCUSSIONWe have investigated the composition and oligomeric statusof two plant small heat shock proteins and followed their subunitexchange reaction in real-time by using mass spectrometry.TaHSP16.9 and PsHSP18.1 have been maintained intactin the gas phase as dodecamers, which is believed to be theirnative form in solution (18, 38). The truncation of up to 13N-terminal amino acids in one PsHSP18.1 monomer does notappear to affect dodecamer formation, which is similar to the

Subunit Exchange of Multimeric Protein Complexes 38927FIG. 6. Decay of homododecamersof PsHSP18.1 and TaHSP16.9 as monitoredby mass spectrometry (cf. Fig.5, summation of relative intensitiesover selected charge states). The fittedlines are for illustrative purposes only.The inset shows a logarithmic plot of thefirst few data points that is used to determinerate constants of the reaction.FIG. 7. Relative concentrations ofthe most abundant heterododecamersas a function of time duringsubunit exchange between PsHSP18.1and TaHSP16.9 at a molar ratio of 3:1(cf. Fig. 5). The fitted lines are for illustrativepurposes only.closely related polydisperse A-crystallins where it has alsobeen shown that truncation of up to 19 amino acids from the Nterminus does not significantly change the subunit organizationof the complex (25).Whereas sHSP complexes appear to be rather rigid stableentities, their structure is in fact very dynamic, enabling themto interact and form heterocomplexes by mutual exchange ofsubunits. With nanoESI MS, we investigated equilibrated mixturesof HSP18.1 and 16.9 at different initial ratios and foundthe distribution of heterocomplexes of varying composition tobe dependent on the initial subunit ratio. Despite the symmetricalwell ordered arrangement of subunits in the dodecamer,we see no indication for preferred stoichiometries. Instead, thesHSP subunits seem to be indistinguishable in the dodecamer,because the end products of the exchange reaction are purelystatistical.Subunit exchange can in principle be thought to proceed viatwo alternative mechanisms: dissociation into smaller subcomplexesfollowed by reassociation into dodecamers or by a “collision/encountercomplex” mechanism. The latter can be ruledout because it would show second-order kinetics. On the otherhand, in the dissociation-association model, the experimentallydetermined first-order kinetics indicate that dissociation ofdodecamers is rate-determining. Subunits constantly dissociatefrom the dodecamer and reassociate. This steady-stateequilibrium is shifted toward the dodecamers with the low levelof subcomplexes limiting the rate of the overall exchange reaction.Consequently, we observe significantly higher proportions

38928Subunit Exchange of Multimeric Protein Complexesof intact complex than dissociation products in the mass spectra(Fig. 2).For the related A-crystallin, it was also found that thedissociation of the intact protein complexes is rate-limiting,suggesting that the oligomers exist in a similar equilibriumwith suboligomeric species (25). A first-order rate constant forsubunit exchange between fluorescence-labeled A-crystallinand unlabeled A- or B-crystallin of 3.8 10 2 min 1 wasreported. By comparison, we determined the rate constants forthe decay of HSP18.1 and 16.9 as 0.20 and 0.24 min 1 , respectively,which is approximately six times faster than the rateconstant for A-crystallin and approximately two orders ofmagnitude faster than that for wild-type transthyretin (42).Because the decay rate constants of HSP18.1 and 16.9 arecomparable, the free enthalpies of activation for the dissociationof the dodecamers into smaller subunits must also be ofsimilar magnitude. Therefore, we believe that the subunit interfacesof the two sHSPs as well as their interactions in thecomplex are closely similar.Approximately 3–4 min after the exchange reaction wasinitiated, only 50% of the homocomplexes remained (Fig. 6),whereas several new heterocomplexes had already formed (Fig.7). The shape of the curves, which go through a maximum, istypical of a sequential reaction, because subsequent exchangereactions only become important after enough intermediatespecies have accumulated. The observed kinetic preference foreven-numbered heterocomplexes indicates that dimeric sHSPsubunits are the predominant exchange units. This finding isin accordance with insight gained from the x-ray structure ofHSP16.9 (Fig. 1) where close interactions among neighboringmonomers have been found, suggesting that dimers are the“building blocks” of the dodecameric protein complex (18).Whereas dimer exchange is faster and thus is clearly favored,the dimeric interaction, however, must at some stage be compromisedbecause heterocomplexes with an uneven number ofsubunits of each kind are also formed. This slower pathwaycould either involve exchange via monomers or proceed viaheterodimers, which were formed in an exchange reaction betweenthe homodimers in solution. After equilibration, thesubunit composition of the dodecamers becomes statistical, nolonger indicating a preference for the exchange of dimers. However,kinetic monitoring of the reaction has revealed dimers asthe predominant units of exchange.Thus, the picture of a very dynamic yet surprisingly stablesHSP complex emerges with dimeric subunits moving in andout of the oligomer on a rapid time scale. This fast associationequilibrium is independent of the presence of other sHSPs butbecomes apparent in the mutual exchange of subunits. Thedynamic nature of the complexes could be essential for theirrole as molecular chaperones, enabling them to associate withnonnative client proteins and shield them from aggregation ashas been suggested for other sHSPs (5). It could also provide amodel for the de novo assembly of the dodecamers from themonomers in the cell. In cases where several different sHSPsare expressed in the same cellular compartment, we can expectthem to readily exchange subunits with other members of thesame class and thus form complexes of mixed composition.Although not much is known regarding the biological relevanceof such sHSP heterocomplexes, it has been found that -crystallinin the human eye lens, which is composed of a 3:1 ratio ofA- to B-crystallin, has greater thermal stability than eitherprotein alone and is therefore more efficient in protecting cellsfrom damage and stress (22). It is also conceivable that theirsubstrate specificity and activity could be moderated by anexchange of subunits with related small heat shock proteins.We have shown that by using mass spectrometry, we caninvestigate the subunit exchange reaction of these two closelyrelated sHSPs and reveal the precise subunit composition ofspecies throughout the entire exchange reaction. 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