Functional Modulation in a Typical Allosteric Protein Revisited - Beyond “T” and “R”

Abstract

The classical tenet of allostery in proteins involves the existence of two alternative but inter-convertible molecular conformations, called T and R, each one exhibiting distinct functional characteristics. Customarily, the T conformation is characterized by a low activity, whereas the R conformation shows high activity. In the textbook case of tetrameric hemoglobin (Hb), ligation at the hemes triggers the structural transition from T to R, with the concomitant functional change from low to high affinity for heme ligands. These two conformations correspond to two distinct structures that differ in the way the two αβ dimers assemble to form an α2β2 tetramer.We have investigated the effects of chemical modification on an allegedly inert interface in Hb, i.e., the α1β1 interface, facing the central cavity, by specific alkylation of residues α104Cys and/or β112Cys, and found that substantial functional changes on Hb do indeed take place. In an extreme case, in the absence of strong heterotropic effectors, one of the derivatives showed a ∼200-fold drop in oxygen affinity and almost abolished cooperativity when compared against its native form. Dimerization degree measured by size exclusion chromatography and isothermal titration calorimetry, as well as 1H-NMR measurements revealed that the permanent-T characteristics for the fully ligated form of modified Hb were not due to the packing of the tetramer into a T conformation, but rather suggested that these characteristics originated from the dimer itself, since the dimerization degree for the fully ligated form was comparable, if not larger than that for the fully ligated native form.These results suggest that a control center for ligand affinity resides within the αβ dimer, and that functional modulation is not necessarily dictated by the tetrameric molecule adopting any of the typical T or R allosteric conformations.