Through a combination of sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation studies, we have established that the pea PBGS protein is largely octameric in the presence of 10 mM MgCl2
). In the absence of added MgCl2
, the enzyme forms hexamers. We find that this process is reversible since adding back MgCl2
restores the octamer assembly.
This system has required careful AU analysis since non-additive oligomeric states, being similar in their molecular masses (220 kDa for the hexamer vs. 293 kDa for the octamer), pose challenges in deconvoluting their contributions to heterogeneity. While SV methods are particularly sensitive to sample heterogeneity, we have found that distinguishing hexamers from octamers in our system is much more challenging and is further exacerbated by the fact that these behave as independent species and thus cannot be probed simply using protein concentration profiling. We did note heterogeneity in the SV samples, using either dCdT+ () or SedFit (analysis not shown), but the species resulting in this heterogeneity were either much smaller than a hexamer/octamer or much larger than an octamer and represent minute (1-2%) amounts. These additional species are likely to reflect either very small amounts of contaminating protein or minor aggregation. Having accounted for these, the fits to the SV data resulted in random residuals. There was no improvement in our ability to resolve hexamers from octamers at the highest speeds attainable with the standard four-hole rotor fitted with Epon centerpieces.
In order to explore this problem more deeply, we created synthetic data to represent a 50:50 mixture of the two species using 10.03 S and 10.94 S as measures of the s-values for the hexamer and octamer, respectively. We also incorporated the typical signal to noise present using an absorbance detection system. In general, we found that the ability of either the dCdT+ or SedFit algorithms to unambiguously assign the fraction of hexamer and octamer present in such a predefined mixture was quite poor since random residuals were obtained from a wide range of hexamer and octamer distributions. A quantitative comparison of the rmsd values for various models, using either the non-interacting discrete species algorithm in Sedfit or the g(s*) multiple species algorithm in dCdT+ is shown in Table S2
(Supplementary Information). In the case of the Sedfit analysis of the synthetic data, we found that we could recover a proportion of 46:54 for the hexamer:octamer species with best-fit s-values of 10.98 S and 10.08 S (while keeping the molecular masses fixed at the theoretical hexamer and octamer values), which compared well to the input values. However, the rmsd of this fit was almost identical to that using other ratios of hexamer and octamer, and more importantly, to an unconstrained single species model. In applying this approach to the pea PBGS data, we found that the rmsd values for a fit to a single species model was identical to that found for different proportions of hexamer and octamer. Similar observations were made using dCdT+. Thus, we conclude that there is significant ambiguity in establishing such heterogeneity in our SV experiments.
The question of hexamer/octamer heterogeneity was better resolved using SE experiments. We were able to see evidence for both hexamers and octamers in the presence and absence of Mg2+, but in each case, one of the components dominated the mixture. Thus, quantifying the contribution of the minor species was difficult. Most problematic was establishing the oligomeric state of the smaller species under octamer-favoring conditions, principally because there was insufficient absorbance to unambiguously establish the assembly state from a limited panel of experimental conditions. This analysis was rendered more difficult because there was no apparent exchange between the hexamer and octamer states, as evidenced by a lack of concentration dependence between the two species. However, careful global analysis of data collected at multiple concentrations and multiple rotor speeds allowed us to establish the presence of these two species unambiguously. The SV and SE experiments, taken in total, have allowed us to elucidate the role of Mg2+ in regulating the distribution of an unusual non-additive mixture of protein assemblies, involving hexamers and octamers.
The role of Mg2+
in the assembly of hexamers and octamers in the pea PGBS is not unprecedented. Metal ions play diverse roles in the majority of PBGS that have been studied. In many cases, Zn2+
binding at the active site is critical for activity (7
). In other cases Mg2+
and some monovalent salts have been shown to be required for activity and are presumed to bind at or near the active site (2
). In addition, many PBGS are regulated through the binding of metal at an allosteric site; these are best characterized as Mg2+
). Plant PBGS enzymes appear to require only Mg2+
for enzymatic activity (2
) and three types of magnesium sites are suggested from the kinetic behavior of pea PBGS (2
). A catalytically essential magnesium (Kd
~ 35 μM) is putatively at the active site. An allosteric magnesium (Kd
~ 2 mM), the location of which is illustrated in , was first observed in the crystal structures of P. aeruginosa
). This site is at a subunit interface that is present in the octameric assembly, but not in the hexameric assembly. It is the existence of this allosteric Mg2+
site that would lead one to ask whether Mg2+
is a structural requirement for formation of the pea PBGS octamer. Prior work showed that removal of Mg2+
with EDTA favors smaller assemblies for the PBGS of both pea and E. coli
). The third magnesium of pea PBGS is inhibitory (Kd
> 10 mM); its location and mechanism are not characterized. The optimal enzyme activity for the chloroplast-located pea PBGS requires Mg2+
concentrations in the physiologically relevant range of 1-10 mM (2
). The current work explores the relationship between Mg2+
and the specific oligomeric assemblies of pea PBGS. We chose to perform the current studies at 10 mM Mg2+
to address the hexamer octamer equilibrium and avoid substantial population of the inhibitory Mg2+
binding site. Here we find that in the absence of substrate, magnesium stabilizes the octameric assembly as predicted from the known location of the allosteric magnesium at a subunit interface that is unique to the octamer.
The existence of non-additive quaternary structure assemblies for PBGS and the realization that a rearrangement between octamer and hexamer constitutes the structural basis for allosteric regulation of plant PBGS by Mg2+
, led us to compare the regulation of PBGS with the classic models of allostery (Monod-Wyman-Changeux vs.
)). These classic models do not consider a situation wherein oligomer dissociation is a required
component of the allosteric mechanism, as it is for PBGS. Hence, we introduced the term morpheein, presented it as a structural basis for allosteric regulation, and suggested that quaternary structure rearrangements similar to that experienced by PBGS may be more common than currently believed (30
). We propose that analytical ultracentrifugation data, fitted to non-additive mixtures or equilibrium models, will assist in the characterization of proteins that can exist as an ensemble of morpheein forms.