In all three of the experimental systems explored in this report, MSSV has proven to be a useful method for the determination of the stoichiometry of proteins that co-sediment as a complex in an SV experiment. In two of these cases (hE3/XDD1 and rTp34/hLF), the stoichiometry established here comports with that measured with another biophysical method, ITC, which is known for its ability to determine the stoichiometries of protein-protein associations [32
In general, the MSSV method demands that at least as many signals be collected as there are proteins in the complex under study. The analyses presented above illustrated two interesting departures from this rule. First, the Zn-rTp34/Zn-hLF interaction was studied with one more signal than thought necessary. The inclusion of three signals (IF, A280, A250) in the analysis afforded spectral resolution whereas an analysis with just two of those signals (IF and A280) failed ( & ). Further analyses (not shown) demonstrate that two signals, IF and A250, would have sufficed. As may be deduced from & , the IF:A250 extinction ratios for the two proteins are significantly different. Most of the spectral discrimination, then, came from the difference in these ratios. However, the inclusion of the A280 data likely added to the hydrodynamic resolution of the experiment, as exclusion of these data would have introduced time gaps wherein no information on the sedimentation of the several species was available. Obviously, if it were known beforehand that the A280 signal would not contribute to the spectral discrimination, the optimal data collection strategy would be to collect only IF and A250 signals.
The other exception to the “one signal per component” rule was in the VCA*/NtA/Arp2/3 study (). It is important to note that this method succeeded only because the molar mass of NtA was a small fraction of that of Arp2/3, and also because only the stoichiometry VCA* and Arp2/3 was monitored. These two conditions must be met before attempting an experiment of this type.
In principle, the MSSV experiment and analysis could accommodate more than the two-protein analyses described above. Given the current capabilities of the Beckman XL-I analytical ultracentrifuge, there is the possibility of spectrally distinguishing four cosedimenting components. One of the signals in such an experiment is necessarily IF. It is possible that the UV-absorbance of proteins would provide the second and third signals (A280 and A250). For most proteins, there is no other convenient peak of UV absorbance; the peptide chain absorbs too strongly in the far UV to be of general use. Consequently, a visible wavelength would be required for a four-signal experiment. Some proteins contain coenzymes that have peaks in the visible region of the spectrum, but most do not. Labeling at least one protein with a chromophore, as was accomplished for VCA in our example, would therefore be required. Importantly, the choices of chromophore and of the position at which to modify the protein are not trivial. If IF is to be used, the chromophore should not absorb the light emitted by that optical system’s laser. It signal increment should provide sufficient signal-to-noise, yet should not overwhelm the capabilities of the on-board spectrophotometer (see above). Further, the site of modification obviously should be distal from the protein’s interaction surface. Our choice of Alexa488 covalently attached to VCA at its amino terminus met all of these criteria.
Of course, there is no reason that only protein-protein interactions can be studied. Any interacting molecule with a measurable signal could be studied. Protein-nucleic-acid interactions seem particularly well suited to the method, as these macromolecules have distinctive UV-absorption signatures. The study of carbohydrates, e.g. in a protein/carbohydrate interaction, should be amenable as long as the size of the carbohydrate and the signal increments of the carbohydrate are known. In such a case, an experiment analogous to those of LeMaire, Salvay, et al
. could be performed [33
]. These researchers were concerned with the amount of detergent bound to their protein, but the same principal holds for protein/carbohydrate interactions.
Previously, other authors have used sedimentation equilibrium (SE) or multisignal sedimentation equilibrium (MSSE) to establish the stoichiometry of protein-protein or protein-nucleic acid interactions [35
]. Two approaches are commonly employed. In the SE approach, several SE experiments are performed with different ratios of the interacting components. In this approach, it is helpful if one of the components dominates the data at one of the signals [37
]. The radial concentration profiles in the centrifugation cells are monitored using a signal that is dominated by one of the components. The several experiments are analyzed individually, and a signal-average buoyant molar mass is derived for each one. A plot of this mass vs. the component ratio should reach a plateau at the buoyant molar mass of the maximal complex. This mass should coincide with the theoretical buoyant molar mass of a complex of a certain stoichiometry, thus establishing that quantity (e.g., see Ucci et al
]). In MSSE, a stoichiometry is assumed, and the data are fit to this experimental model. The goodness of the fit is taken as confirmation of the stoichiometry [35
]. Often in such analyses, other information about the associating proteins is known. This information may be built into the analysis; for example, the buoyant molar masses of the components may be known, or the number of components and their approximate buoyant molar mass may be known from an SV experiment. MSSV represents a complementary approach to the problem with distinct advantages: (a) while an SE experiment generally takes days to perform, MSSV can be done in hours (overnight); (b) SE and MSSE do not give the experimenter information regarding the hydrodynamic properties (scomplex
) of the complex, while MSSV does; (c) the data basis of the MSSV experiment is significantly larger than SE or MSSE, which may lead to better spectral resolution of species [1
]; and (d) SE data analysis requires that an interaction model be imposed, while MSSV is model-free in that regard. It should be noted that short-column SE experiments may be performed in hours [37
], mitigating point (a) above, at the expense of making the data basis of those experiments smaller. Therefore, SE, MSSE, or MSSV may be used to determine stoichiometry; for a quick, accurate determination of stoichiometry only, a single MSSV experiment should suffice.
As pointed out by Balbo et al
], MSSV is best suited to experimental systems that have a slow koff
relative to the time taken to perform an SV experiment (koff
). By simulating data for fast interactions, they found that they may be characterized by MSSV, but one of the components must be present at a large molar excess over the other. For the hE3/XDD1 and Zn-rTp34/Zn-hLF systems, the koff
’s are likely to be slow; they do not dissociate when subjected to size-exclusion chromatography (not shown). Further, the molar excesses used in these experiments ensure a high degree of occupation of the complex. The koff
of the VCA/Arp2/3 [9
] interaction is fast by the above criterion, but the presence of large molar excesses of this protein ensures full occupation in our experiments.
In conclusion, MSSV has proved to be a dependable method to determine the stoichiometry of proteins in a hetero-associating complex. In addition to the experiments presented above, several other groups (e.g., see [1
]) have successfully used this technique. The multisignal approach has also been used to characterize protein/detergent complexes [34
]. MSSV adds a new tool to those already available to the biophysicist to answer one of the fundamental questions that arises from studying protein-protein interactions in detail, and should be applicable to a wide variety of experimental systems.