Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of strong to ultratight protein interactions using differential scanning calorimetry

Publisher Summary This chapter discusses the molecular properties and catalytic properties of E. coli alkaline phosphatase. Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three brands, four and five equally spaced bands have been found. Single gene mutations apparently affect each isozyme, for the mobility of all the bands are affected without altering the spacing. Levinthal concluded that all of the isozymes were coded for by the same gene. Studies have obtained large single crystals of E. coli alkaline phosphatase. Initial X-ray studies by Hanson show the crystals to be of the space group P3121, each unit cell containing three dimers. The unit cell dimensions are a = 70.5 A, b = 70.5 A, c = 155.6 A, β = 120°, which is consistent with the globular form predicted by the hydrodynamic frictional ratio.

17 E. coli Alkaline Phosphatase

Zinc proteins are an integral component of the proteome of all domains of life. Zn(II), one of the most widespread transition elements, serves multiple functions in proteins, such as a catalytic co-factor, a structural center and a signaling component. The mechanism by which proteins associate with and dissociate from Zn(II) and the factors that modulate their affinity and stability remain incompletely understood. In this article, we aim to address how zinc binding sites present in proteins differ in their architecture and how their structural arrangement is associated with protein function, thermodynamic and kinetic stability, reactivity, as well as zinc-dependent regulation. Here, we emphasize that the concentration-dependent functionality of the interprotein zinc binding site may serve as another factor regulating the relationship between cellular Zn(II) availability and protein function.

Relationship between the architecture of zinc coordination and zinc binding affinity in proteins – insights into zinc regulation

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.

Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding.

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

The binding of Zn2+ to Escherichia coli alkaline phosphatase has been studied by pH titrations and equilibrium dialysis using 1,10-phenanthroline as competing chelating agent.



Measurements of the strength of binding under different experimental conditions have been performed:



1
At constant pH with different concentrations of chelating agent.

2
At different pH with constant concentration of chelating agent.

3
In the presence of denaturing agent at pH 8.0.





It was found on the basis of equilibrium dialysis experiments that the binding of the two zinc atoms to alkaline phosphatase may be described as coordination to the two equivalent and independent sites.



The high values of the binding constants, and the fact that denaturation destroys the specific binding of zinc to alkaline phosphatase indicates chelate formation with the protein, unless some unknown prosthetic groups are involved.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb19590.x

H. Csopak

Papers

The Specific Binding of Zinc(II) to Alkaline Phosphatase of Escherichia coli