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The contribution of the α1β1half‐oxygenated tetramer [αβ:αO2βO2] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O2‐driven protein structure changes, was measured as 21ΔGc = 5.1 ± 0.3 kcal for the Zn/FeO2 analog. For the Fe/FeCN analog, 21ΔGc was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Hb cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly‐ligated species, providing strong support for the previously determined consensus partition function of O2 binding and for the Symmetry Rule model of hemoglobin cooperativity (Ackers et al., Science 1992;255:54–63). Cooperativity of normal human hemoglobin is shown to depend on site‐configuration, and not solely the number of O2 bound, nor the occupancy of α vs. β subunits. Verification of a unique contribution from the α1β1doubly‐oxygenated species to the equilibrium O2 binding curve strongly reinforces the Symmetry Rule interpretation that the α1β1dimer acts both as a structural and functional element in cooperative O2 binding. Proteins 2000;41:23–43. © 2000 Wiley‐Liss, Inc.

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].

No abstract

The cro repressor from bacteriophage lambda is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer. Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding. A method is described by which cro repressor can be labeled in vivo with [(35)S]methionine to a specific activity of 2 x 10(15) cpm/mol. As a prelude to binding studies, the association equilibrium of cro was determined over the range 10(-)(9)-10(-)(3) M using large-zone analytical gel chromatography with radiolabeled repressor. The data are best described by a monomer-dimer stoichiometry with an equilibrium constant of 3.07 (+/-1.08) x 10(6) M(-)(1) total cro monomer. Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients. Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 100 microg/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.