AFFINImeter: A software to analyze molecular recognition processes from experimental data

Abstract

The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software.Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of supramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

“…The integrated heat data generated a binding isotherm with a single inflection point, and a mild slope ( Figure 2 A, right panel and Table 1 ). The fit of the obtained data, performed using the AFFINImeter software [ 49 ] and a model involving a single set of sites, showed that two Ni(II) ions bind per Hp UreG monomer with similar affinity ( K d = 72 µM), a favorable enthalpic contribution and a minor entropic impact ( Table 1 ). Previously reported studies on Hp UreG mutants indicated that at least one Ni(II) binding site is located on the CPH motif [ 20 , 29 ].…”

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

“…Displacement ITC experiments were performed to measure arginine binding in S69A; in this case, a sample of protein 100 μ m + histidine 4 m m was titrated with arginine 4 m m . Data were analyzed using the software Affinimeter . K d , the enthalpy change (∆ H ), and the binding stoichiometry ( n ) were determined by nonlinear fitting of the normalized titration data based on a 1 : 1 binding model (Fig.…”

Lewis Acidic Boranes, Lewis Bases, and Equilibrium Constants: A Reliable Scaffold for a Quantitative Lewis Acidity/Basicity Scale

“…The resulting heat signals were integrated to give the heats per injection. The correlation of the heats per injection with the molar ratio of Lewis base andb orane gave ab inding isotherm (Figure 2c), which was analyzed with a1 :1 interaction model, [15] to derive the associationc onstants, K B .T hreei ndividual ITC experiments for each Lewis acid/Lewis base combination were performed. These individual resultsw ere averaged to determine the equilibriumc onstant, K B .T hus, ITC allowed equilibrium constants to be reliably determined in the range of 10 3 < K B < 10 7 m À1 .H owever,o ur ITC instrument could not be operated under completely inert conditions, and side reactions with traces of moisture werealimitation for boranes more acidic than 1e.…”