Chapter 4
Kinetic Methods of Deducing Binding Mechanisms Involving
Intrinsically Disordered Proteins
Elin Karlsson and Per Jemth
Abstract
There are multiple examples of protein–protein interactions involving one intrinsically disordered protein
region binding to an ordered protein domain in a coupled binding and folding reaction. Similarly to protein
folding studies, much effort has been devoted to understanding the mechanisms of such coupled binding
and folding reactions. In this chapter, we describe how kinetics can be used to assess binding mechanisms
with focus on fluorescence-monitored stopped-flow experiments. The approach can be applied more
generally to any protein interaction with or without a coupled conformational change and to other kinetic
techniques. Determining binding mechanisms is a great challenge and while “proving” a mechanism may
be futile, it is possible to deduce the simplest scenarios, which are consistent with experimental data.
Key words Binding mechanism, Intrinsically disordered proteins, Kinetics, Stopped flow
1 Introduction
1.1 Intrinsically
Disordered Proteins
A large fraction of the proteome contains regions that are disordered, i.e., amino acid sequences that do not fold into well-defined
compact structures but remain highly dynamic under physiological
conditions [1]. These intrinsically disordered proteins (IDPs) could
contain transient structure, and they may have short unfolded
regions or disorder which may be a property the whole protein.
Disordered regions can contain interaction motifs that bind to
folded protein domains. Upon binding, the disordered region
usually adopts a well-defined conformation, but there are examples
where disorder prevails in the protein–protein complex [2]. In
other cases, the disordered regions flanking the binding motifs
modulate binding through short-lived attractive or unfavorable
repulsive interactions within the complex [3]. Interactions involving IDPs are being increasingly identified using bioinformatics as
well as in structural studies and so this mode of binding must be
advantageous. For example, signal transduction pathways and the
transcriptional machinery provide multiple examples of such
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_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
105
Kinetic Methods of Deducing Binding Mechanisms Involving
Intrinsically Disordered Proteins
Elin Karlsson and Per Jemth
Abstract
There are multiple examples of protein–protein interactions involving one intrinsically disordered protein
region binding to an ordered protein domain in a coupled binding and folding reaction. Similarly to protein
folding studies, much effort has been devoted to understanding the mechanisms of such coupled binding
and folding reactions. In this chapter, we describe how kinetics can be used to assess binding mechanisms
with focus on fluorescence-monitored stopped-flow experiments. The approach can be applied more
generally to any protein interaction with or without a coupled conformational change and to other kinetic
techniques. Determining binding mechanisms is a great challenge and while “proving” a mechanism may
be futile, it is possible to deduce the simplest scenarios, which are consistent with experimental data.
Key words Binding mechanism, Intrinsically disordered proteins, Kinetics, Stopped flow
1 Introduction
1.1 Intrinsically
Disordered Proteins
A large fraction of the proteome contains regions that are disordered, i.e., amino acid sequences that do not fold into well-defined
compact structures but remain highly dynamic under physiological
conditions [1]. These intrinsically disordered proteins (IDPs) could
contain transient structure, and they may have short unfolded
regions or disorder which may be a property the whole protein.
Disordered regions can contain interaction motifs that bind to
folded protein domains. Upon binding, the disordered region
usually adopts a well-defined conformation, but there are examples
where disorder prevails in the protein–protein complex [2]. In
other cases, the disordered regions flanking the binding motifs
modulate binding through short-lived attractive or unfavorable
repulsive interactions within the complex [3]. Interactions involving IDPs are being increasingly identified using bioinformatics as
well as in structural studies and so this mode of binding must be
advantageous. For example, signal transduction pathways and the
transcriptional machinery provide multiple examples of such
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_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
105