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G K Ackers

Publications and source records attributed to G K Ackers.

At least 19 recordsLinked to original sources
PubMed1992

Cooperative oxygen binding, subunit assembly, and sulfhydryl reaction kinetics of the eight cyanomet intermediate ligation states of human hemoglobin.

Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Gel↗
PubMed1992

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.

Allosteric Regulation↗
PubMed1992

Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin.

The inability to localize and measure the free energy of protein structure and structure change severely limits protein structure-function investigations. The local unfolding model for protein hydrogen exchange quantitatively related the free energy of local structural stability with the hydrogen exchange rate of concerted sets of structurally related protons. In tests with a number of modified hemoglobin forms, the loss in structural free energy obtained locally from hydrogen exchange results matches the loss in allosteric free energy measured globally by oxygen-binding and subunit dissociation experiments.

Allosteric Regulation↗
PubMed1992

Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.

Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Molecular code for cooperativity in hemoglobin.

Although tetrameric hemoglobin has been studied extensively as a prototype for understanding mechanisms of allosteric regulation, the functional and structural properties of its eight intermediate ligation forms have remained elusive. Recent experiments on the energetics of cooperativity of these intermediates, along with assignments of their quaternary structures, have revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule. This "symmetry rule" translates the configurational isomers of heme-site ligation into six observed switchpoints of quaternary transition. Cooperativity arises from both "concerted" quaternary switching and "sequential" modulation of binding within each quaternary form, T and R. Binding affinity is regulated through a hierarchical code of tertiary-quaternary coupling that includes the classical allosteric models as limiting cases.

Allosteric Regulation↗
PubMed1992

Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

Hemoglobins↗
PubMed1992

Regulation of oxygen affinity by quaternary enhancement: does hemoglobin Ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.

Allosteric Regulation↗
PubMed1992

Weighting functions and parameter resolvability for oxygenation data subject to error in the independent variable.

Parameter resolvability and bias has been investigated for weighted nonlinear regression of data where the independent variable is subject to instrumental uncertainty. The specific example of cooperative oxygenation of hemoglobin was studied, where fractional saturation is determined spectrophotometrically and the oxygen activity is measured with a Clark polarographic electrode. For this system the instrumental uncertainty in the oxygen electrode was measured directly and the influence of the uncertainties on resolution of oxygen binding parameters was determined by Monte Carlo simulations. Four weighting functions were tested for their ability to minimize parameter uncertainty and bias: (1) uniform weighting; (2) "propagated weighting" whereby uncertainties in the independent variable are propagated into and added to uncertainties of the dependent variable; (3) Hill plot transform, or "end weighting"; and (4) maximum likelihood analysis, where deviations between fitting function and data are minimized as weighted horizontal and vertical distance vectors. Results of the Monte Carlo simulations favor the use of either uniform weighting, propagated weighting, or maximum likelihood weighting methods. Use of the Hill transform as a weighting function produced poorer parameter resolvability and inaccurate representation of the data in general. Bias error was negligible for all weighting functions.

Electrodes↗
PubMed1991

Energetics of subunit dimerization in bacteriophage lambda cI repressor: linkage to protons, temperature, and KCl.

A common feature of gene regulatory systems is the linkage between reversible protein oligomerization and DNA binding. Experimental dissection using temperature dependence of the subunit-subunit energetics and their linkage to processes such as ion binding and release is necessary for characterization of the chemical forces that contribute to cooperativity and site specificity. We have therefore studied the effects of temperature, proton activity, and monovalent salt on monomer-dimer assembly of the lambda cI repressor using a recently developed gel chromatographic procedure. This technique has made possible studies in the previously inaccessible picomolar concentration ranges where the assembly reactions occur. Upon formation of the dimer interface in the range pH 5-9, we find an overall absorption of protons which is temperature-dependent. The dimerization reaction displays a large negative enthalpy of association at all conditions studied (pH 5, 7, and 9). The reaction is also dependent on monovalent salt concentration: subunit association is weaker at low-salt conditions. The results suggest that a repulsive interaction between negatively charged side chains (i.e., aspartates and glutamates) on each monomer surface is attenuated by increasing concentrations of KCl. Formation of the dimer interface may be mediated by absorption of cations which stabilize the complex.

Bacteriophage lambda↗
PubMed1991

Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR.

The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.

Bacteriophage lambda↗
PubMed1991

Experimental resolution of cooperative free energies for the ten ligation species of cobalt(II)/iron(II)-CO hemoglobin.

Cooperative free energies have been determined for the 10 ligation species of human hemoglobin in the Co(II)/Fe(II)-CO system. In this system, subunits containing unligated cobaltous hemes coexist in the same tetramer with naturally occurring ferrous hemes that are ligated with carbon monoxide. Tetramers comprising the 10 structurally unique combinations of ligated and unligated subunits were characterized in terms of their dimer-tetramer assembly free energies. By use of the thermodynamic linkage between assembly and ligation, the experimentally resolved values were used to obtain the corresponding cooperative free energies (i.e., the differences between actual free energies of ligation and the summed contributions of intrinsic values). The results obtained are in general accord with previous findings on this same system (Imai et al., 1980). The present study extends this earlier work by resolving the cooperative properties of each configurational isomer of the doubly ligated tetramers. The 10 ligation species were found to distribute into 5 discrete cooperative free energy levels according to a combinatorial code which includes, as a special case, the code found previously with cyanomethemoglobin and manganese-substituted hemoglobin (Smith et al., 1987; Daugherty et al., 1991). This distribution exhibits additional characteristics found in the oxygenation of normal ferrous hemoglobin including the quaternary enhancement effect (Mills & Ackers, 1979a,b). These results, and those of the following paper (Doyle et al., 1991), strongly support the premise that a common set of qualitative rules governs the cooperative interactions in hemoglobin irrespective of

Carbon Monoxide↗
PubMed1991

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin.

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobins↗
PubMed1991

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching--i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the alpha 1 beta 2 intersubunit contact. These studies also reveal significant cooperativity within each alpha 1 beta 1 dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.

Allosteric Site↗
PubMed1991

Quantitative study of protein association at picomolar concentrations: the lambda phage cl repressor.

A method has been developed for radiolabeling the lambda cl repressor to a specific activity sufficiently high to permit accurate quantitation of the protein in the picomolar range of concentration. Procedures are described whereby the labeled protein can be used for accurate quantitative study of the energetics of repressor assembly by large zone analytical gel chromatography. This methodology is applicable to other systems in which the stoichiometry and energetics of tightly associating DNA binding proteins are currently difficult to measure.

Chromatography, Gel↗
PubMed1990

Direct and indirect pathways of functional coupling in human hemoglobin are revealed by quantitative low-temperature isoelectric focusing of mutant hybrids.

Functional energetic coupling within human hemoglobin has been explored by using quantitative analysis of asymmetric mutant hybrid equilibria. Previous work showed that the free energy of cooperativity is largely attributable to alterations in free energy that accompany changing interactions at the interface between alpha 1 beta 1 and alpha 2 beta 2 dimers [Pettigrew et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1849]. However, the issue of how cooperativity-linked sites in the molecule are energetically coupled in manifesting cooperative ligation is still not well delineated. In this paper we address the questions of what types of functional coupling pathways are operational in hemoglobin, what some of their characteristics are, and how they are related to one another. By constructing asymmetric mutant hybrid hemoglobins, we can assay how two structurally identical, symmetrically equivalent sites are energetically coupled in manifesting subunit assembly and/or cooperative ligation. Asymmetric hybrid hemoglobins, i.e., those containing a single modified site, cannot be isolated and must be studied in equilibrium with their symmetric parent molecules. In order to study these asymmetric hybrid equilibria, we have developed new theory and quantitation techniques to augment the low-temperature quenching and isoelectric focusing procedures of Perrella et al. [(1978) Anal. Biochem. 88, 212]. Studies of these mutant hybrid hemoglobins have provided evidence for three distinct types of energetic coupling within the hemoglobin tetramer. All alpha 1 beta 2 interface sites examined are involved in cooperativity-linked indirect coupling. Within the context of this indirect "pathway" there exist two different types of direct long-range coupling. One of these classes of direct long-range pathways is linked to cooperative ligand binding while the other class is not.

Cold Temperature↗
PubMed1990

Analysis of hemoglobin oxygenation from combined equilibrium and kinetic data. Is quaternary enhancement necessary?

An experimental approach based on four independent techniques, in which kinetic and equilibrium measurements of subunit assembly reactions are combined with concentration-dependent oxygen-binding curves, has previously been used to resolve parameters of the linkage system for human hemoglobin over a wide range of conditions [(G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A. 71 (1974) 4312; F.C. Mills et al., Biochemistry 15 (1976) 1093; M.L. Johnson et al., Biochemistry 15 (1976) 5363). Throughout this extensive body of results it has been found that the affinity for binding oxygen to tetramers at the fourth step exceeds the mean affinity of dissociated dimers. The existence of this "quaternary enhancement" effect has recently been questioned by Gibson and Edelstein (J. Biol. Chem. 262 (1987) 516) and by Philo and Lary (J. Biol. Chem. 265 (1990) 139) on the basis of kinetically derived oxygen-binding constants that do not exhibit quaternary enhancement. These authors have also suggested that quaternary enhancement might not be necessary to explain the oxygen-binding data mentioned above. In this study, we have explored the effect of constraining the numerical analysis of oxygen-binding data against the new kinetically derived binding constants. It is found that the sets of linkage constants which are compatible with both the oxygen-binding data and the new kinetically derived dimer binding constant require both quaternary enhancement and substantial dimer cooperativity. Increasing the dimer cooperativity to compensate completely for quaternary enhancement requires both dimeric and tetrameric binding constants that disagree with the kinetically derived values. Thus, the quaternary enhancement effect cannot be eliminated by readjustment of the remaining constants of the linkage system. Possible sources of the discrepancy between the kinetically derived binding constants and the otherwise self-consistent data from the other four techniques are discussed.

Hemoglobins↗
PubMed1990

The energetics of ligand-linked subunit assembly in hemoglobin require a third allosteric structure.

For partially ligated cyanomet hemoglobins, Smith and Ackers (Proc. Natl. Acad. Sci. U.S.A. 71 (1985) 4312) determined the free energies of dimer-tetramer assembly for all of the partially ligated species using a combination of kinetic and equilibrium methods. They found a third apparent cooperative free energy level in addition to those of deoxy- and cyanomethemoglobin. Using cryogenic methods, Perrella et al. (Biophys. Chem. 35 (1990) 97) confirmed the existence of the third cooperative free energy level, but found a different energy level assignment for one of the species. These combined studies have yielded a solid data base for considering mechanistic issues. The number of cooperative free energies delta Gc can, in principle, be different from the number of molecular forms which have unique free energies of heme-heme interaction, since delta Gc can be an average over conformational subspecies. Furthermore, since the delta Gc values are determined from free energies of dimer-tetramer assembly, it is necessary to evaluate possible contributions from dimeric properties, and from quaternary constraint (or enhancement) effects associated with subunit assembly. In this paper we analyze the observed distributions of apparent delta Gc values among the various ligation states in terms of mechanisms based on two interconvertible molecular forms (R and T) under the most general conditions in which (i) dimers may be cooperative, (ii) ligand affinities of alpha-subunits may be different within tetramers and dimers, and the same for beta-subunit affinities, and (iii) dimers need not be halves of R-state tetramers. It is found that the experimental distributions are inconsistent with even the most general model of the two-state class; thus, at least three molecular forms of tetramer are required, each with an individually different value of cooperative free energy (heme-heme interaction). This result implies the existence of at least three corresponding molecular structures; while a degeneracy of multiple structures into only a few dominant free energy levels is frequently to be expected, the reverse situation is extremely unlikely.

Allosteric Regulation↗