Aspects of protein-DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors, IPTG and the reporter gene lacZ.

Protein-DNA interactions are central to the control of gene expression across all forms of life. The development of approaches to rigorously model such interactions has often been hindered both by a lack of quantitative binding data and by the difficulty in accounting for parameters relevant to the intracellular situation, such as DNA looping and thermodynamic non-ideality. Here, we review these considerations by developing a thermodynamically based mathematical model that attempts to simulate the functioning of an Escherichia coli expression system incorporating two of the best characterised prokaryotic DNA binding proteins, Lac repressor and lambda CI repressor.

Kinetic trapping of a key hemoglobin intermediate.

The complete binding cascade of human hemoglobin consists of a series of partially ligated intermediates. The individual intermediate binding constants cannot be distinguished in O(2) binding curves, however, each constant can be determined from the O(2)-induced change in assembly constant for the α(2)β(2) tetramer from its constituent αβ dimers. The characterization of these O(2) binding constants has shown the Hb cascade to be asymmetric in nature, with binding dependent upon the specific distribution of O(2) among the four hemesites.

The Hill coefficient: inadequate resolution of cooperativity in human hemoglobin.

The Hill coefficient nH is a dimensionless parameter that has long been used as a measure of the extent of cooperativity. Originally derived from the oxygen-binding curve of human hemoglobin (Hb) by A. V.

Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 2. Stepwise cooperative free energy.

Stepwise cooperative free energies and intermediate Hill coefficients are used to assess the presence of noncooperative sequences in the database of binding free energies previously obtained for the eight partially ligated intermediates of human hemoglobin, encompassing a variety of hemesite analog substitutions. This analysis is prompted by the observed noncooperative binding of two ligands to hemoglobins that are partially substituted with Zn2+-heme, an analog of deoxy Fe2+-heme (Holt et al. (2005) Biochemistry 44, XXXXX).

Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 1. Cooperative and noncooperative oxygen binding in Zn-substituted hemoglobin.

The complete binding cascade of human hemoglobin consists of eight partially ligated intermediates and 16 binding constants. Each intermediate binding constant can be evaluated via dimer-tetramer assembly when ligand configurations within the tetramer are fixed through the use of hemesite analogs. The Zn/Fe analog, in which the nonbinding Zn2+ heme substitutes for deoxy Fe2+ heme, also permits direct measurement of O2 binding to the remaining Fe2+ hemesites within the symmetrically ligated Hb tetramers.

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 2. Cooperative free energies of (alphaFeCObetaFe)2 and (alphaFebetaFeCO)2 T-state tetramers.

Ligand photodissociation experiments are used to measure the prephotolysis equilibria between doubly liganded R and T quaternary conformers of the symmetric Fe-Co HbCO hybrids, (alpha(FeCO)beta(Co))(2) and (alpha(Co)beta(FeCO))(2). The free energies obtained from these data are used to calculate the cooperative free energies of the (alpha(FeCO)beta(Fe))(2) and (alpha(Fe)beta(FeCO))(2) intermediate CO-ligation states of normal hemoglobin in the T conformation, quantities important to the evaluation of current models of cooperativity. The symmetry rule model, incorporating sequential cooperativity of T-state ligand binding within an alphabeta dimer in addition to the traditional two-state cooperativity of the tetramer, predicts a larger free energy penalty for disturbing both dimers in a doubly liganded T tetramer than would be expected in the two-state model as currently formulated.

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 1. Microstate linear free energy relations.

A novel model linking the thermodynamics and kinetics of hemoglobin's allosteric (R --> T) and ligand binding reactions is applied to photolysis data for human HbCO. To describe hemoglobin's kinetics at the microscopic level of structural transitions and ligand-binding events for individual [ij]-ligation microstates ((ij)R --> (ij)T, (ij)R + CO --> ((i)(+1))(k)R, and (ij)T + CO --> ((i)(+1))(k)T), the model calculates activation energies, (ij)DeltaG(++), from previously measured cooperative free energies of the equilibrium microstates (Huang, Y., and Ackers, G.

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 alpha beta 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.

Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies.

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin alpha(1)beta(1)half-oxygenated intermediate supports the symmetry rule model of allosteric regulation.

The contribution of the alpha(1)beta(1)half-oxygenated tetramer [alphabeta:alphaO(2)betaO(2)] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O(2)-driven protein structure changes, was measured as (21)DeltaG(c) = 5.1 +/- 0.

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined.

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations.

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.

Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli.

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the alpha and beta chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes.

Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis.

Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive. In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity.

A quantitative cryogenic gel-shift technique for analysis of protein-DNA binding.

A cryogenic gel mobility shift technique was developed in which a mixture of protein and DNA samples at equilibrium is rapidly quenched and electrophoresed at -40 degrees C. The rapid and sustained drop in temperature results in almost complete stabilization of the equilibrium species distribution. Autoradiogram analysis of relative abundances for the bound and free DNA sites is carried out over a range of initial binding ratios to yield the binding curve and equilibrium constant as in the usual gel-shift assay.

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants.

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.

Hydropathic analysis of the non-covalent interactions between molecular subunits of structurally characterized hemoglobins.

The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results.