Analytical ultracentrifugation in structural biology

Abstract

Researchers in the field of structural biology, especially X-ray crystallography and protein nuclear magnetic resonance, are interested in knowing as much as possible about the state of their target protein in solution. Not only is this knowledge relevant to studies of biological function, it also facilitates determination of a protein structure using homogeneous monodisperse protein samples. A researcher faced with a new protein to study will have many questions even after that protein has been purified. Analytical ultracentrifugation (AUC) can provide all of this information readily from a small sample in a non-destructive way, without the need for labeling, enabling structure determination experiments without any wasting time and material on uncharacterized samples. In this article, I use examples to illustrate how AUC can contribute to protein structural analysis. Integrating information from a variety of biophysical experimental methods, such as X-ray crystallography, small angle X-ray scattering, electrospray ionization-mass spectrometry, AUC allows a more complete understanding of the structure and function of biomacromolecules.

Keywords: AUC; Crystallography; Interaction; Protein.

Fig. 1

Distribution of sedimentation coefficients c(s_20,w) for the general transcription factor TFIIE. Calculated c(s_20,w) is plotted against the sedimentation coefficient s_20,w. Experiments were conducted at an initial protein concentration of 1.37 mg/mL in 20 mM sodium phosphate (pH 7.9), 500 mM NaCl, 10 mM 2-mercaptoethanol, 10% glycerol. Data were collected at a rotor speed of 40,000 rpm and at time intervals of 5 min. The calculated values of the weight-average s_20,w and frictional ratio (f/fo) are 3.3 S and 2.1, respectively

Fig. 2

Schematic drawing of 11-mer trp RNA-binding attenuation protein (TRAP) and trimer anti-TRAP (AT) interaction. Wild-type Bacillus stearothermophilus TRAP (PDB ID  1QAW; Chen et al. 1999), Bacillus subtilis anti-TRAP (PDB ID 2BX9; Shevtsov et al. 2005)

Fig. 3

Sedimentation velocity analysis of mixtures of wild-type Bacillus stearothemophilus 11-mer TRAP and AT. The TRAP concentration was fixed at 0. 5 mg/ml, and varying amounts of AT were added. The data obtained from six experiments are overlaid. The molar ratios (by monomers) of TRAP-AT are 1:0 (blue), 1:0.5 (green), 1:1 (light green), 1:2 (brown), 1:6 (purple), 1:10 (red)

Fig. 4

Comparison of TRAP complexes. Wild-type TRAP-AT complex (PDB ID 2ZP8); TRAP–RNA complex (PDB ID 1C9S) (Antson et al. 1999)