Gary K. Ackers

[9] Quantitative DNase footprint titration: A method for studying protein-DNA interactions

Publisher Summary This chapter discusses that individual-site binding isotherms are uniquely suited to permit the resolution of interaction parameters for systems exhibiting cooperative interactions between multiple sites. The analysis is completely general. Any number of specific sites can be analyzed regardless of the nature of the cooperative or anticooperative interactions among them. The development of the footprint titration method, which permits resolution of individual-site isotherms, permits quantitative characterization of systems that act as critical regulators of gene transcription. The thermodynamic parameters that are resolved from footprint titration can be used in two important ways to further the understanding of gene regulation. First, the binding affinities of the various components of a gene regulatory system can be used to deduce the mechanism of the regulation—for example, the successful modeling of the switch from the lysogenic-to-lytic growth stage of the lambda phage. Second, the range of precisely controlled experimental conditions over which the technique is applicable allows to measure other thermodynamic parameters—for example, enthalpies and entropies—to study the roles of the various noncovalent forces of interaction involved in protein-DNA binding and site recognition.

Free energy coupling within macromolecules: The chemical work of ligand binding at the individual sites in co-operative systems

Individual-site binding curves such as those obtainable from techniques of DNase footprinting or nuclear magnetic resonance spectroscopy can be used to monitor structurally localized events within biopolymers. This paper discusses thermodynamic aspects of individual-site ligand binding for co-operative systems where the binding of ligand at a local site is coupled to binding of the same ligand species at other sites within the macromolecule. Individual-site binding isotherms have the following properties. (1) They provide a direct indication of the role played by the particular site in the overall binding reaction. (2) They can be used to determine the energetic contribution of loading the site regardless of the complexity of the system. (3) They can be used to resolve microscopic equilibrium constants and co-operativity constants in cases where the classical isotherm is incapable of such resolution. The microscopic constants bear a complex relation to the chemical work of loading each individual site. For a system with two interacting sites we derive analytical relationships between the individual-site loading energies and the microscopic constants. These relationships prescribe, for any values of the microscopic constants, how the co-operative energy is partitioned between events at the two sites. At fixed ligand activity the binding free energy can be estimated directly from an individual-site isotherm. This quantity, which is also a composite of the microscopic constants, provides a useful measure of site--site interaction. Several examples and applications are discussed for these properties of individual-site binding reactions.

[9] Study of protein subunit association equilibria by elution gel chromatography

Publisher Summary This chapter describes methods for determining the stoichiometries and equilibrium constants for a self-associating protein by elution gel chromatography. The technique of elution gel chromatography is based upon the molecular size-dependent penetration of solute molecules into porous networks of gels—such as crosslinked dextrans (Sephadex), polyacrylamide, and agaroses. The same considerations are also applied to the columns of controlled-pore glass. In general, the range of molecular sizes for complexes that can be analyzed by the chromatography methods described in the chapter lies between several angstroms and several hundred angstroms in molecular diameter, as a correspondingly wide range of gel porosities is readily available. The selected aspects of theory pertaining to elution chromatography are briefly described in the chapter in an attempt to present a coherent framework. Different aspects of elution chromatography on associating solutes are also presented in the chapter with specific details on the equipment and procedures, which may be utilized for optimum experimental design. The most common gel chromatography technique is the elution experiment in which a concentration–volume profile is measured for the sample emerging at the bottom of a column. A wide variety of assays may be performed (enzymatic, radioactive, etc.) if fractions are collected of the eluted solute. However, the chapter is concerned only with the most generally useful approach of continuous spectrophotometric monitoring.

[37] Measurement and analysis of ligand-linked subunit dissociation equilibria in human hemoglobins

Publisher Summary This chapter describes methods that are useful in the studies of reversible association–dissociation equilibria in human hemoglobin and focuses primarily on the problems of category. The methods described are successfully applied to the study of (1) the linkage between dimer–tetramer association and oxygen binding, (2) the effects of temperature, pH, and chloride on the oxygenation linked dimer–tetramer reactions, and (3) the ligand-linked self-association of isolated α and β chains. The chapter presents basic definitions and concepts of the ligand-linked dissociation scheme for human hemoglobin. The chapter also describes some experimental techniques, such as equilibrium gel permeation methods for determining equilibrium constants of subunit association and the haptoglobin-binding method for determining tetramer to dimer dissociation rates in unliganded hemoglobins. The reversible dissociation of human hemoglobin tetramers occurs in the region of neutral pH leading to the formation of dimers.

Linked functions in allosteric proteins: Extension of the concerted (MWC) model for ligand-linked subunit assembly and its application to human hemoglobins

Abstract The allosteric model of Monod et al. (1965) (MWC) has been extended to take into account the effects of subunit dissociation. The problem is formulated theoretically in terms of a general model for two allosteric species (dimers and tetramers) linked by a polymerization reaction. Relationships are presented for interpreting the dimer-tetramer association constants in terms of allosteric model parameters. Sub-cases of the general model were tested against recent experimental data on the oxygenation-linked dimer-tetramer equilibria in normal human hemoglobin and in the variant hemoglobin Kansas (β102, Asp → Thr). The objectives of these analyses were: (1) to find the simplest models capable of describing the linked dimer-tetramer equilibria in the two hemoglobin systems, and (2) to evaluate the corresponding model parameters so that allosteric properties of the two hemoglobins may be compared. In the simplest version of the model, the dimer is half of an R-state tetramer. This model was found to be excluded unequivocally by the data for both normal hemoglobin and hemoglobin Kansas when the α and β chains have equal binding affinities. When this two-state model was modified to permit non-equivalent affinities for the chains, the model could be fitted to hemoglobin Kansas, but not to hemoglobin A. A model, in which the dimers are allowed to exist in a state different from the tetramer R state, was found to be consistent with the data for hemoglobin A, with equivalent binding by the α and β chains. For hemoglobin A, the unliganded R-state tetramers have a different subunit dissociation energy from that of fully liganded R-state tetramers. The simplest model capable of describing both hemoglobin A and hemoglobin Kansas was obtained by extending this three-state model to permit (but not require) functional non-equivalence of the α and β chains. For these MWC models, unique estimates were obtained for the model parameters. The allosteric constants for tetrameric hemoglobins A and Kansas are approximately equal. The value obtained from hemoglobin A is similar to previous estimates, whereas the value for hemoglobin Kansas is lower than previously estimated (Edelstein, 1971) by approximately two orders of magnitude. The low affinity of hemoglobin Kansas tetramer does not arise from an unusually high allosteric constant favoring the T-state species. It is largely the consequence of a greatly reduced oxygen affinity of β chains in the T state, and reduced values for the ratio between affinities in the R and T states.

Engineering and design of blood substitutes

Clinical experiences with chemically modified and genetically engineered hemoglobin blood substitutes have uncovered new side-effects that must be addressed before a viable oxygen-carrying alternative to blood can be developed. These include, among others, vasoactivity and rapid autoxidation resulting in free-radical-mediated toxicity. Research is now being directed towards understanding the mechanisms of these toxic side-effects and developing methods of overcoming them.

Quantitative study of protein association at picomolar concentrations: The λ phage cl repressor

Abstract A method has been developed for radiolabeling the λ 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.

[15] Studies of protein ligand binding by gel permeation techniques

Publisher Summary This chapter discusses experimental methods that utilize the principles of gel permeation for the study of macromolecule–ligand interactions. Several procedures have been developed in recent years that take advantage of the equilibrium dialysis properties of gel partitioning systems for the study of ligand binding. The simplest method in terms of required equipment is the Hummel–Dreyer technique carried out by elution chromatography. The only equipment required is a chromatographic column, a small amount of gel, and a spectrophotometric monitoring device. Such activity assays permit the study of enzyme systems in impure form and may extend the concentration range down into the nanogram per milliliter region. A generally more precise technique is the Brumbaugh–Ackers, which utilizes direct optical column scanning and measures ligand binding at many points within a single column. Although this approach requires considerably more sophisticated instrumentation and expense, the advantages of speed and precision certainly warrant the additional investment for any extensive studies of macromolecular interaction.

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin ?1?1half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

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.

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of αβ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer–dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both α- or both β-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer–dimer contacts is not communicated across the dimer–dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T/R) component.