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