Chapter 18
Characterization of Protein–Nucleic Acid Complexes
by Size-Exclusion Chromatography Coupled with Light
Scattering, Absorbance, and Refractive Index Detectors
Ewa Folta-Stogniew
Abstract
Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS)
enables determination of the molecular weight, oligomeric state, and stoichiometry of protein–nucleic acid
complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a
challenge in determination of molecular weight and stoichiometry based solely on the elution position
from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape
of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the
complex between the reverse transcriptase (RT) domain from group II intron–maturase from Eubacterium
rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between
single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting
repressor (FIR).
Key words SEC/MALS, Laser light scattering, Size-exclusion chromatography (SEC), Molecular
weight, Protein–nucleic acid complex, Stoichiometry, Oligomeric state
1 Introduction
Light scattering is a spectroscopic technique for determination of
the molecular weight of biopolymers in solution. Typically, molecular weight of protein complexes is estimated from size-exclusion
chromatography (SEC) or from analytical ultracentrifugation
(AUC); both techniques are limited by the influence of the shape
of the complex on the measurement. Similarly to sedimentation in
the analytical ultracentrifuge (AUC), light scattering measurement
is an absolute method for determination of molecular weight and
does not rely on reference standards.
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_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
381
Characterization of Protein–Nucleic Acid Complexes
by Size-Exclusion Chromatography Coupled with Light
Scattering, Absorbance, and Refractive Index Detectors
Ewa Folta-Stogniew
Abstract
Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS)
enables determination of the molecular weight, oligomeric state, and stoichiometry of protein–nucleic acid
complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a
challenge in determination of molecular weight and stoichiometry based solely on the elution position
from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape
of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the
complex between the reverse transcriptase (RT) domain from group II intron–maturase from Eubacterium
rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between
single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting
repressor (FIR).
Key words SEC/MALS, Laser light scattering, Size-exclusion chromatography (SEC), Molecular
weight, Protein–nucleic acid complex, Stoichiometry, Oligomeric state
1 Introduction
Light scattering is a spectroscopic technique for determination of
the molecular weight of biopolymers in solution. Typically, molecular weight of protein complexes is estimated from size-exclusion
chromatography (SEC) or from analytical ultracentrifugation
(AUC); both techniques are limited by the influence of the shape
of the complex on the measurement. Similarly to sedimentation in
the analytical ultracentrifuge (AUC), light scattering measurement
is an absolute method for determination of molecular weight and
does not rely on reference standards.
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_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
381