Chapter 12
A Quick Primer in Fluorescence-Based Equilibrium
and Pre-steady State Methods for Determining
Protein–Nucleotide Affinities
Harland E. Brandon and Hans-Joachim Wieden
Abstract
Biomolecular interactions facilitate the biochemical processes that sustain life. Proteins, RNAs, and ribonucleoprotein complexes perform cellular functions that range from catalyzing the formation or cleavage of
bonds to being structural building blocks, both of which are only possible through the interaction with
their respective biomolecular partner(s). Having access to the parameters that describe these interactions is
important for our understanding of the principles that underlie enzymatic and nonenzymatic processes.
Here we describe two fluorescence-based approaches to determine two key parameters, the affinity and the
rate of association/dissociation of a protein and a ligand. Considerations are provided to expand the
described approach to other experimental systems.
Key words Affinity, Fluorescence, Stopped-flow, Equilibrium binding, Pre-steady state, Nucleotide,
GTPase, FRET
1 Introduction
Biological systems are composed of many biomolecules that are
required to interact with each other to drive the chemical processes
that facilitate life as we know it. Understanding which and how
biomolecules interact can provide insight into the mechanism that
are at the core of biochemical processes and how they form the
molecular pathways that underlie cellular life. Proteins make up a
large percentage of the total dry weight of a cell, with 55% of a
single Escherichia coli cell being protein [1]. Proteins are constantly
interacting with various ligands in the cell, some that they act upon,
and others that they only transiently interact with. Knowing the
affinity of a protein to a particular ligand informs our understanding of how the protein functions mechanistically and how it integrates into the complex reaction pathways of the cell. Nucleotides,
the ligand of interest in the following examples, are the building
blocks of nucleic acids, along with being important secondary
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
273
A Quick Primer in Fluorescence-Based Equilibrium
and Pre-steady State Methods for Determining
Protein–Nucleotide Affinities
Harland E. Brandon and Hans-Joachim Wieden
Abstract
Biomolecular interactions facilitate the biochemical processes that sustain life. Proteins, RNAs, and ribonucleoprotein complexes perform cellular functions that range from catalyzing the formation or cleavage of
bonds to being structural building blocks, both of which are only possible through the interaction with
their respective biomolecular partner(s). Having access to the parameters that describe these interactions is
important for our understanding of the principles that underlie enzymatic and nonenzymatic processes.
Here we describe two fluorescence-based approaches to determine two key parameters, the affinity and the
rate of association/dissociation of a protein and a ligand. Considerations are provided to expand the
described approach to other experimental systems.
Key words Affinity, Fluorescence, Stopped-flow, Equilibrium binding, Pre-steady state, Nucleotide,
GTPase, FRET
1 Introduction
Biological systems are composed of many biomolecules that are
required to interact with each other to drive the chemical processes
that facilitate life as we know it. Understanding which and how
biomolecules interact can provide insight into the mechanism that
are at the core of biochemical processes and how they form the
molecular pathways that underlie cellular life. Proteins make up a
large percentage of the total dry weight of a cell, with 55% of a
single Escherichia coli cell being protein [1]. Proteins are constantly
interacting with various ligands in the cell, some that they act upon,
and others that they only transiently interact with. Knowing the
affinity of a protein to a particular ligand informs our understanding of how the protein functions mechanistically and how it integrates into the complex reaction pathways of the cell. Nucleotides,
the ligand of interest in the following examples, are the building
blocks of nucleic acids, along with being important secondary
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
273