Conformational equilibrium of demetalized concanavalin A

Abstract

Concanavalin A (Con A) is known to exist in two conformations [Brown, R. D., III, Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883-3896] that differ in their metal ion and saccharide binding properties. The conformation that binds metal ions tightly, and which is associated with saccharide binding, has been designated as "locked" and that which binds metal ions only weakly as "unlocked". In the presence of excess metal ions, such as Mn2+ and Ca2+, essentially 100% of the protein is in the locked conformation. The scheme proposed to explain these effects [Koenig, S. H., Brewer, C. F., & Brown, R. D., III (1978) Biochemistry 17, 4251-4260] predicts an equilibrium between these conformations for the apoprotein. By monitoring the solvent proton relaxation dispersion as equimolar concentrations of Mn2+ and Ca2+ are titrated, at 5 degrees C, into an apo-Con A solution that had been equilibrated at 25 degrees C, we find that 12.5% of the apoprotein is in the locked conformation, corresponding to an energy separation of 1.2 kcal mol-1. We also show that these conformations can be separated by column chromatography at 5 degrees C and that the 100% unlocked form prepared in this way returns to the expected equilibrium mixture when kept at 25 degrees C.