Self crowding of globular proteins studied by small-angle x-ray scattering

Area of Science:

• Biophysics
• Structural Biology
• Physical Chemistry

Background:

• Proteins in solution exhibit complex behaviors at high concentrations.
• Understanding these interactions is crucial for various biological and biotechnological applications.
• Small-angle X-ray scattering (SAXS) is a powerful technique for probing macromolecular structure and interactions.

Purpose of the Study:

• To investigate the solution behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at high concentrations using SAXS.
• To evaluate the applicability of the hard-sphere fluid model in describing interparticle interference effects.
• To explore the influence of buffer composition on protein-protein interactions.

Main Methods:

• High-concentration SAXS measurements were performed on Mb and BPTI in solutions containing D2O and urea.
• Experimental scattering profiles were analyzed by comparison with theoretical models, including crystal structures and a hard-sphere fluid model.
• Second virial coefficients were estimated to quantify interparticle interactions.

Main Results:

• Significant interparticle interference effects were observed for both proteins, indicating nonideal behavior.
• The hard-sphere model accurately described Mb behavior with a radius close to its structural estimate.
• BPTI scattering showed less interference than predicted by the hard-sphere model, with buffer-dependent variations suggesting electrostatic influences.

Conclusions:

• The hard-sphere model is a useful tool for analyzing SAXS data from concentrated protein solutions, particularly for well-behaved proteins like Mb.
• BPTI's behavior is more complex, influenced by both repulsive and attractive intermolecular forces, with buffer choice playing a significant role.
• Phosphate ions may mediate attractive interactions in BPTI solutions, distinct from stable oligomer formation.