assessing relative dissociation constants for different RNA substrates prior to complex formation for crystallization experiments.
3.8.2 Biophysical
Characterization of RNA–
Protein Interactions
Biophysical characterization methods, e.g., SPR and NMR, can be
used to confirm and characterize RNA–protein interactions. SPR
represents a popular technique for determining kinetic rate constants (k a and k d ) and equilibrium constants (K d ) based on mass
concentration-dependent changes in refractive index at the sensor
chip surface that are monitored in real time. Protein or RNA can be
immobilized on the sensor chip surface to detect and monitor
RNA–protein interactions. In addition, NMR can be used to determine kinetic parameters and derive structural information. SPR and
NMR are covered in more detail elsewhere in this book (see Chapters 11 and 17).
3.9 Forming
the RNA–Protein
Complex
In principle, RNA–protein complexes can be prepared simply by
mixing the RNA and protein constituents at the desired ratios and
setting up crystallization trials. Alternatively, the individual components of the complex can be mixed at lower concentrations, with
the complex subsequently purified using gel filtration chromatography and concentrated prior to setting up the crystallization
experiments. For in vitro assembly of large multi-protein complexes, the individual components may need to be added in a
specific order, with the assembly of the complex being monitored
using native gel electrophoresis and mass spectrometry. Typically,
the preferred method is to add RNA directly to the protein sample
owing to the high cost of synthesized RNA. K d is a measure of
affinity between ligand and protein. K d ¼ [P][L]/[PL] where [P],
[L], and [PL] correspond to the molar concentrations of protein,
ligand, and the protein–ligand complex, respectively. For RNA–
protein complexes, K d represents the concentration of RNA at
which the RNA binding site is half occupied, i.e., [PL] ¼ [P]. To
ensure 90% fractional saturation of the RNA binding site, the free
RNA concentration must be in excess of the free protein concentration and, at equilibrium, should not deplete to less than 10 Â K d .
In practice, the high RNA concentrations used in RNA–protein
complex crystallography experiments usually saturate the protein–
RNA-binding site. A molar ratio of 1:0.9 RNA:protein is recommended as a starting point for initial crystallization screening [18].
3.10 Setting Up
RNA–Protein
Crystallization
Experiments
The typical RNA–protein complex crystallography cascade is presented in Fig. 5. The first hurdle is obtaining well-ordered, diffraction quality crystals. The protein must be pure and monodisperse,
and the RNA–protein complex must be homogeneous and should
not noticeably degrade over the time course of the crystallization
experiment. The most routine method for setting up crystallization
experiments is vapor diffusion using hanging or sitting drops.
Crystallization trials are usually carried out using protein
Studying RNA–Protein Complexes Using X-Ray Crystallography
433
3.8.2 Biophysical
Characterization of RNA–
Protein Interactions
Biophysical characterization methods, e.g., SPR and NMR, can be
used to confirm and characterize RNA–protein interactions. SPR
represents a popular technique for determining kinetic rate constants (k a and k d ) and equilibrium constants (K d ) based on mass
concentration-dependent changes in refractive index at the sensor
chip surface that are monitored in real time. Protein or RNA can be
immobilized on the sensor chip surface to detect and monitor
RNA–protein interactions. In addition, NMR can be used to determine kinetic parameters and derive structural information. SPR and
NMR are covered in more detail elsewhere in this book (see Chapters 11 and 17).
3.9 Forming
the RNA–Protein
Complex
In principle, RNA–protein complexes can be prepared simply by
mixing the RNA and protein constituents at the desired ratios and
setting up crystallization trials. Alternatively, the individual components of the complex can be mixed at lower concentrations, with
the complex subsequently purified using gel filtration chromatography and concentrated prior to setting up the crystallization
experiments. For in vitro assembly of large multi-protein complexes, the individual components may need to be added in a
specific order, with the assembly of the complex being monitored
using native gel electrophoresis and mass spectrometry. Typically,
the preferred method is to add RNA directly to the protein sample
owing to the high cost of synthesized RNA. K d is a measure of
affinity between ligand and protein. K d ¼ [P][L]/[PL] where [P],
[L], and [PL] correspond to the molar concentrations of protein,
ligand, and the protein–ligand complex, respectively. For RNA–
protein complexes, K d represents the concentration of RNA at
which the RNA binding site is half occupied, i.e., [PL] ¼ [P]. To
ensure 90% fractional saturation of the RNA binding site, the free
RNA concentration must be in excess of the free protein concentration and, at equilibrium, should not deplete to less than 10 Â K d .
In practice, the high RNA concentrations used in RNA–protein
complex crystallography experiments usually saturate the protein–
RNA-binding site. A molar ratio of 1:0.9 RNA:protein is recommended as a starting point for initial crystallization screening [18].
3.10 Setting Up
RNA–Protein
Crystallization
Experiments
The typical RNA–protein complex crystallography cascade is presented in Fig. 5. The first hurdle is obtaining well-ordered, diffraction quality crystals. The protein must be pure and monodisperse,
and the RNA–protein complex must be homogeneous and should
not noticeably degrade over the time course of the crystallization
experiment. The most routine method for setting up crystallization
experiments is vapor diffusion using hanging or sitting drops.
Crystallization trials are usually carried out using protein
Studying RNA–Protein Complexes Using X-Ray Crystallography
433