TABLE 1.

Protein behaviors that might indicate an equilibrium of morpheein forms.

Characteristic	Comments
SDS-pure protein separating into alternate bands on native PAGE can indicate multimers of alternate stoichiometry (or conformation).	Native PAGE can be routinely incorporated into the final stages of protein characterization.
SDS-pure protein that separates into alternate forms using ion exchange chromatography	This has been used to monitor the interconversion of alternate assemblies as a function of ligand.
The enzyme-kinetic phenomenon known as hysteresis.	This can be observed if the transition from a low activity form to a high activity form occurs during the assay [as we have shown for the R240A variant of PBGS (Tang et al., 2006].
Double hyperbolic kinetics (the sum of two hyperbola with different kinetic constants).	This can indicate alternate morpheein forms with different kinetic constants. Seeing this may require using a broad range of substrate concentration (see Figure 4).
X-ray crystal structures of multi-domain proteins that cannot be superposed without clashes. This observation is often dismissed as an artifact of crystal packing.	Many multi-domain proteins do not produce diffraction quality crystals. A common approach is to truncate one or more domains. If overlaying the common elements of such structures causes domains to clash, this could be a sign of alternate assemblies. This was observed for alternate truncated constructs of HIV integrase (Andrake and Skalka, 2015).
Protein concentration dependent specific activity can indicate alternate activities associated with different multimeric stoichiometries.	This is seen for all PBGS that use only magnesium [e.g., (Petrovich et al., 1996; Kervinen et al., 2000; Shanmugam et al., 2010)].
Evidence for soluble protein multimers that dissociate along a hydrophilic protein-protein interface.	This is a difficult characteristic to search for as many crystal structure files are at insufficient resolution to position water molecules. PBGS crystal structures contain phylogenetically conserved water molecules at the subunit-subunit interfaces that dissociate upon formation of the dimeric morpheein forms (Selwood et al., 2008).