Identification of an allosterically sensitive unfolding unit in hemoglobin

Abstract

Hydrogen-exchange studies locate a set of seven allosterically sensitive amide NH protons side by side around two turns of the F-FG helical segment in the hemoglobin beta chain. Some of these protons are on the aqueous protein surface and some deeply inside, yet they all exchange with solvent protons at similar rates. Further, they move in unison to a new common rate when hemoglobin changes its allosteric form. These observations and analogous results for other proteins appear to be inconsistent with penetration-dependent models which relate H-exchange rate to solvent accessibility in the native state. Rather, these results point to sizeable fluctuational distortions that make small sets of protons more or less equally accessible in some transient H-exchange transition state, as visualized in the local unfolding model. The set of allosterically sensitive protons studied here exchanges 30-fold faster in liganded hemoglobin than in the deoxy form. In terms of the unfolding model, this means that the F-FG structure is relatively destabilized in oxyhemoglobin, so that the allosterically linked change in structural free energy at F-FG favors the deoxy state. The 30-fold change in H-exchange rate suggests a contribution to the allosteric free energy by this segment of 2 kcal (1 cal = 4.184 J). These experiments utilized a labeling technique, described earlier, that selectively places tritium on sites whose H-exchange rates are sensitive to the protein functional state, and used a method introduced by Rosa & Richards (1979,1981) to locate this label in the protein. The latter method, which rapidly separates protein fragments under conditions that can preserve exchangeable label, was here brought to a more quantitative level. Taken together, these techniques provide a "functional labeling" method capable of selectively labeling and identifying protein segments that participate in functional interactions.