Chapter 19
Analytical Ultracentrifugation for Analysis
of Protein–Nucleic Acid Interactions
Andrea Bogutzki and Ute Curth
Abstract
Analytical ultracentrifugation is a powerful tool to characterize interactions of macromolecules in solution.
In sedimentation velocity experiments, the sedimentation of interaction partners and complexes can be
monitored directly and can be used to characterize interactions quantitatively. As an example, we show how
the interaction of the clamp loader subcomplex of DNA polymerase III from E. coli and a template/primer
DNA saturated with single-stranded DNA-binding protein can be analyzed by analytical ultracentrifugation
with fluorescence detection.
Key words Analytical ultracentrifugation, Fluorescence detection, Sedimentation velocity experiments, Protein–DNA interaction, DNA replication, Single-stranded DNA-binding protein (SSB),
Clamp loader of DNA polymerase III, Sortase-mediated specific protein-labeling
1 Introduction
1.1 Analytical
Ultracentrifugation
In analytical ultracentrifugation (AUC), macromolecules in the
absence and presence of their interaction partners can be exposed
to a high gravitational field in solution and their sedimentation can
be traced by real-time detection. As a result, not only can information about the size, the shape, and the molar mass of the molecules
be obtained, but in the case of interacting systems, information
about the stoichiometry and the stability of the complex can also
be received. There are two generally different methods used in
AUC: sedimentation equilibrium and sedimentation velocity
experiments. Whereas the former one can be used to determine
the molar mass of molecules and complexes independent of their
shape, the latter one primarily reveals information about the size
and the shape of the particles. Although both methods can be used
to quantitatively characterize macromolecular interactions, we will
focus here on sedimentation velocity experiments and their application in the investigation of protein–DNA interactions. Basic
principles, recent developments, and applications of analytical
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_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
397
Analytical Ultracentrifugation for Analysis
of Protein–Nucleic Acid Interactions
Andrea Bogutzki and Ute Curth
Abstract
Analytical ultracentrifugation is a powerful tool to characterize interactions of macromolecules in solution.
In sedimentation velocity experiments, the sedimentation of interaction partners and complexes can be
monitored directly and can be used to characterize interactions quantitatively. As an example, we show how
the interaction of the clamp loader subcomplex of DNA polymerase III from E. coli and a template/primer
DNA saturated with single-stranded DNA-binding protein can be analyzed by analytical ultracentrifugation
with fluorescence detection.
Key words Analytical ultracentrifugation, Fluorescence detection, Sedimentation velocity experiments, Protein–DNA interaction, DNA replication, Single-stranded DNA-binding protein (SSB),
Clamp loader of DNA polymerase III, Sortase-mediated specific protein-labeling
1 Introduction
1.1 Analytical
Ultracentrifugation
In analytical ultracentrifugation (AUC), macromolecules in the
absence and presence of their interaction partners can be exposed
to a high gravitational field in solution and their sedimentation can
be traced by real-time detection. As a result, not only can information about the size, the shape, and the molar mass of the molecules
be obtained, but in the case of interacting systems, information
about the stoichiometry and the stability of the complex can also
be received. There are two generally different methods used in
AUC: sedimentation equilibrium and sedimentation velocity
experiments. Whereas the former one can be used to determine
the molar mass of molecules and complexes independent of their
shape, the latter one primarily reveals information about the size
and the shape of the particles. Although both methods can be used
to quantitatively characterize macromolecular interactions, we will
focus here on sedimentation velocity experiments and their application in the investigation of protein–DNA interactions. Basic
principles, recent developments, and applications of analytical
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_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
397