Signal and binding. II. Converting physico-chemical responses to macromolecule-ligand interactions into thermodynamic binding isotherms.
Physico-chemical titration techniques are the most commonly used methods in characterizing molecular interactions. These methods are mainly based on spectroscopic, calorimetric, hydrodynamic, etc., measurements. However, truly quantitative physico-chemical methods are absolutely based on the determination of the relationship between the measured signal and the total average degree of binding in order to obtain meaningful interaction parameters. The relationship between the observed physico-chemical signal of whatever nature and the degree of binding must be determined and not assumed, based on some ad hoc intuitive relationship/model, leading to determination of the true binding isotherm. The quantitative methods reviewed and discussed here allow an experimenter to rigorously determine the degree of binding and the free ligand concentration, i.e., they lead to the construction of the thermodynamic binding isotherm in a model-independent fashion from physico-chemical titration curves.
Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation.
A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.
The Contemporary British History Play
Introduction Biographical History Plays Social History Plays Oppositional History Plays Marxist and Socialist History Plays Feminist History Plays Deconstructionist and Postmodern History Plays and Satire The Search for a Theatrical Form Appendix A: Performance Dates of Contemporary British History Plays Appendix B: Performance Locales of Contemporary British History Plays Bibliography Index