Single‐site modifications of half‐ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer
The patterns of energetic response elicited by single‐site hemoglobin mutations and chemical mocdifications have been determined in order to probe the dimer–dimer interface of the half‐ligated tetramer (species) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules1. In this study the free energies of quaternary assembly(dimers to tetramers) were determined for aseries of 24 tetrameric species in which one dimeric half‐molecule is ligated while the adjacent αβ dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half‐ligated tetramers and the unligated tetramers over the entire spatial distrubution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer–dimer interface of the half‐ligated tetramer(species) has the same quaternary sturcture as that of the unligated molecule, i.e, “quaternary T.” This quaternary structure assignment of species  strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T → R quaternary switchpoints2. Analysis of the observed Symmetry Rule behaviour in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between α and β subunits of the dimeric half‐tetramer within quaternary T. The mutational results indicate that these interactions are not “paid for” by breaking or making noncovalent bonds at the dimer–dimer interface (α1β2). They arise from structural and energetic changes that are “internal” to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of “tertiary constraint” is thus generated by the first binding step and is propagated to the second hemesite while the dimer–dimer interface α1β2serves as a constraint. The “sequential” cooperativity that occurs within the half‐molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers. When the constraint is released functionally by T to R dimer rearrangement (at each switch‐point specified by the a Symmetry Rule) the alterations of interfacial bonds then dominate the energetics of cooperativity. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.
Publication Source (Journal or Book title)
Proteins: Structure, Function, and Bioinformatics
LiCata, V., Dalessio, P., & Ackers, G. (1993). Single‐site modifications of half‐ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer. Proteins: Structure, Function, and Bioinformatics, 17 (3), 279-296. https://doi.org/10.1002/prot.340170306