Chapter 3
Interactions of a Signal Transduction Protein Investigated
by Fluorescence Stopped-Flow Kinetics
Stephen R. Martin and Maria J. Schilstra
Abstract
To understand cellular processes such as biochemical pathways and signaling networks, we need to
understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational
modifications, or other mechanisms. To do so, we break these systems down into their elementary steps,
which are almost invariably either unimolecular or bimolecular reactions that frequently occur on
sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing
in less than 2 ms, are generally suitable for the study of such reactions. The application of these techniques
to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals
of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis,
1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the
pre-steady state) under single turnover conditions.
The individual steps in complex biochemical reaction schemes determine how fast systems can respond to
incoming signals and adapt to changed conditions [1, 2]. This chapter is concerned with in vitro techniques
that have been developed to study fast reactions in solution, and we present the study of various interactions
of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for
understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.
Key words Calmodulin, Kinetics, Rate constants, Fluorescence, Stopped-flow, Data analysis and
modeling
1 Introduction
1.1 The Biological
System Under Study
Changes in calcium concentration act as a ubiquitous intracellular
signal responsible for controlling many biological processes such as
contraction, secretion, fertilization, and cell proliferation [3]. In all
eukaryotic cells, one of the important proteins that mediates Ca
2+
signaling is calmodulin (CaM). Upon Ca
2+ stimulation, CaM binds
to and modulates the activity of a diverse number of enzymes,
including a family of CaM-dependent serine/threonine protein
kinases [4]. CaM is a 148-amino acid protein whose crystal
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
83
Interactions of a Signal Transduction Protein Investigated
by Fluorescence Stopped-Flow Kinetics
Stephen R. Martin and Maria J. Schilstra
Abstract
To understand cellular processes such as biochemical pathways and signaling networks, we need to
understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational
modifications, or other mechanisms. To do so, we break these systems down into their elementary steps,
which are almost invariably either unimolecular or bimolecular reactions that frequently occur on
sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing
in less than 2 ms, are generally suitable for the study of such reactions. The application of these techniques
to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals
of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis,
1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the
pre-steady state) under single turnover conditions.
The individual steps in complex biochemical reaction schemes determine how fast systems can respond to
incoming signals and adapt to changed conditions [1, 2]. This chapter is concerned with in vitro techniques
that have been developed to study fast reactions in solution, and we present the study of various interactions
of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for
understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.
Key words Calmodulin, Kinetics, Rate constants, Fluorescence, Stopped-flow, Data analysis and
modeling
1 Introduction
1.1 The Biological
System Under Study
Changes in calcium concentration act as a ubiquitous intracellular
signal responsible for controlling many biological processes such as
contraction, secretion, fertilization, and cell proliferation [3]. In all
eukaryotic cells, one of the important proteins that mediates Ca
2+
signaling is calmodulin (CaM). Upon Ca
2+ stimulation, CaM binds
to and modulates the activity of a diverse number of enzymes,
including a family of CaM-dependent serine/threonine protein
kinases [4]. CaM is a 148-amino acid protein whose crystal
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
83