Subunit Exchange of Multimeric Protein Complexes

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