Simultaneous determination of thermodynamic and kinetic data by isothermal titration calorimetry
Graphical abstract
Introduction
Much work has gone into the development of Isothermal titration calorimetry (ITC) [1,2]. Nowadays it is an indispensable tool for the parameterization of biomolecular recognition events. Titration calorimetry is the only technique that enables simultaneous, direct determination of the Gibbs free energy change ΔG and enthalpy change ΔH and calculation of the entropy change ΔS of a binding reaction using the following equation:
ITC has become a routine tool in the design of novel drug candidates as it provides information about the thermodynamic quantities governing the protein-ligand binding event [3]. Correlating ITC data with structural information from macromolecular crystallography can then support the understanding of the origin of enthalpy-entropy compensation in a given protein-ligand system. Furthermore, it provides information about the changes in the thermodynamic characteristics evolving within a series of congeneric compounds, which result from e.g. non-optimally positioned polar functionalities [4]. It is well appreciated that thermodynamics only provide one view of a ligand's affinity. Another facet is contributed by binding kinetics. Nowadays, it is well accepted that kinetics are equally important for the conversion of a ligand into a drug candidate, as they often yield a better estimation of in vivo efficacy than thermodynamics or affinity per se [5].
The development of the novel concept called kinITC complements ITC by providing the association (kon) and dissociation rate constants (koff) for a 1:1 binding model, which are linked to Kd by Eq. (2) [[6], [7], [8]]. Kd and koff are determined by the titration experiment, kon is calculated using Eq. (2).
kinITC as implemented in the AFFINImeter suite thus provides rapid access to both thermodynamic and kinetic data for a full characterization of the fashion in which a ligand's affinity for its target is conveyed [6]. Zihlmann et al. [9] exemplarily succeeded in demonstrating that kinITC could be used to fully characterize inhibitors of the bacterial adhesin FimH. Additionally, these findings enabled the assignment of the role of sugar moieties and aglycone residues for the kinetic binding profiles of the inhibitors.
Section snippets
Design of titration protocols
There are many set screws to an ITC experiment. The concentrations of protein (cp) and ligand (cl), the number of injections (Ni), the volume of injections (Vi), the injection time (ti), the delay between consecutive injections (ts), the stirring speed (vr), and data collection filter time constant are all independent experimental variables chosen by the operator. These variables determine the dependent experimental variables, the molar ratio of titrant to titrand (rm) at each data point, the
Thermodynamic results
Fig. 2 shows thermodynamic results for titration experiments of the same protein-ligand system with the different protocols (Tables S1–S2).
All protocols yield comparable dissociation constants within the margin of error although the mixed protocol suggests a deviation from the other protocols. While the mixed protocol shows a notable difference between average and globally fitted value, these are virtually the same for the other protocols, respectively.
Furthermore, the standard protocol shows a
Comparison with surface plasmon resonance
Nowadays, surface plasmon resonance (SPR) is the method of choice for the measurement of biomolecular reaction kinetics. Thermodynamically, ITC and SPR seem to provide the same values for a given protein-ligand system using the same materials as demonstrated in a study with bovine CAII and CBS [23]. However, ITC and SPR differ fundamentally with respect to the state of the protein, which is either freely floating in solution or immobilized on an SPR chip via a linker. As Dumas et al. [25] and
Conclusion
It has been shown that a combination of the standard protocol and a thermodynamically optimized protocol with varying injection volumes can be used to extract both thermodynamic and kinetic information for 1:1 protein-ligand reactions from one single titration experiment with kinITC with lower uncertainty in the thermodynamic and kinetic quantities than from the standard protocol with the caveat, that injection speed must be maintained for experiments whose results are to be compared later.
Setup of measurement procedure
For establishing a titration protocol, optimized for kinetic data collection, the following aspects should be considered. For practical considerations of the experimental execution see Velázquez Campoy and Freire [27]. The AFFINImeter kinITC software can approximately determine rate constants in the ranges of 5·103 < kon < 8.4·105 and 6.5·10−4 < koff < 1.2·10−1 [28].
- 1.
Determine the active fraction of the protein (i.e. protein capable of binding the ligand) and the ligand's affinity for it by an
Credit roles
Steffen Glöckner: Performed the measurements, optimized and developed the protocol, performed data analysis & curation, wrote the first draft of the manuscript, visualized the results, project administration.
Gerhard Klebe: Funding acquisition, project administration, made the necessary resources available, supervised the study, helped in writing and editing the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Dr. Khang Ngo for HPLC analysis of CBS. We thank AFFINImeter for scientific consulting. We thank Malvern Panalytical for the allocation of a PEAQ-ITC instrument to our laboratory and its maintenance.
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