Chapter 2
A Familiar Protein–Ligand Interaction Revisited
with Multiple Methods
Xiaochun Li-Blatter, Ludovit Zweifel, and Timothy Sharpe
Abstract
The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been wellcharacterized by biophysical methods and structural biology. In this chapter, we present a series of
experiments designed to detect and quantify that interaction using several commonly available biophysical
methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.
These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of
representative data from several years of practical courses, we are able to demonstrate the robustness of the
protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the
degree of consistency between those methods when applied to a simple system in a single location by
different experimenters.
Key words Dissociation constant, Teaching, Orthogonal assay, Thermal shift, Fluorescence, Microscale thermophoresis, Isothermal titration calorimetry, Surface plasmon resonance, Global fitting
1 Introduction
1.1 Several
Techniques, One
Experimental System
This chapter presents a series of experiments using different techniques to detect binding and to measure the dissociation constant
(K d ) for the interaction of the trisaccharide tri-N-acetyl glucosamine (NAG3) with the protein hen egg white lysozyme
(HEWL). The idea to develop set experiments to measure the
same interaction using different methods arose in the context of a
biophysics core facility, where there is a regular need to train people
to use instruments, and to troubleshoot and benchmark instruments after malfunction and repair.
This demands a simple and robust experimental system, with
reagents that are readily available, easy to handle, and relatively
inexpensive. The binding of NAG3 to HEWL seemed a good
candidate system, and a series of experiments were developed over
a few years, starting with ITC (see Note 1) and expanding to
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
47
A Familiar Protein–Ligand Interaction Revisited
with Multiple Methods
Xiaochun Li-Blatter, Ludovit Zweifel, and Timothy Sharpe
Abstract
The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been wellcharacterized by biophysical methods and structural biology. In this chapter, we present a series of
experiments designed to detect and quantify that interaction using several commonly available biophysical
methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.
These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of
representative data from several years of practical courses, we are able to demonstrate the robustness of the
protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the
degree of consistency between those methods when applied to a simple system in a single location by
different experimenters.
Key words Dissociation constant, Teaching, Orthogonal assay, Thermal shift, Fluorescence, Microscale thermophoresis, Isothermal titration calorimetry, Surface plasmon resonance, Global fitting
1 Introduction
1.1 Several
Techniques, One
Experimental System
This chapter presents a series of experiments using different techniques to detect binding and to measure the dissociation constant
(K d ) for the interaction of the trisaccharide tri-N-acetyl glucosamine (NAG3) with the protein hen egg white lysozyme
(HEWL). The idea to develop set experiments to measure the
same interaction using different methods arose in the context of a
biophysics core facility, where there is a regular need to train people
to use instruments, and to troubleshoot and benchmark instruments after malfunction and repair.
This demands a simple and robust experimental system, with
reagents that are readily available, easy to handle, and relatively
inexpensive. The binding of NAG3 to HEWL seemed a good
candidate system, and a series of experiments were developed over
a few years, starting with ITC (see Note 1) and expanding to
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
47