Protein interactions in concentrated ribonuclease solutions

• Published in: Journal of crystal growth Vol. 196; no. 2; pp. 185 - 192
• Main Authors: Boyer, Mireille, Roy, Marie-Odile, Jullien, Magali, Bonneté, Françoise, Tardieu, Annette
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.1999; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biochemistry, Molecular Biology; Biological and medical sciences; Biophysics; Chemical Physics; Crystallization; Dynamic light scattering; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Life Sciences; Physics; Protein interactions; Proteins; Ribonuclease A; Small angle X-ray scattering; 61.10.Eq; Ribonuclease A; 78.35.+c; Dynamic light scattering; Crystallization; 87.15.Da; Protein interactions; Small angle X-ray scattering; Hydrolases; Molecular interaction; Esterases; Pancreatic ribonuclease; Enzyme
• ISSN: 0022-0248; 1873-5002
• DOI: 10.1016/S0022-0248(98)00838-0

To investigate the protein interactions involved in the crystallization process of ribonuclease A, dynamic light scattering (DLS) and small angle X-ray scattering experiments (SAXS) were performed on concentrated solutions. Whereas the translational diffusion coefficient obtained from DLS is sensitive to thermodynamic and hydrodynamic interactions and permits to calculate an interaction parameter, the shape of the SAXS curves is related to the type of interaction (attractive or repulsive). We compared the effect of pH on protein interactions in the case of two types of crystallizing agents: a mixture of salts (3 M sodium chloride plus 0.2 M ammonium sulfate) and an organic solvent (ethanol). The results show that in the presence of ethanol, as in low salt, protein interactions become more attractive as the pH increases from 4 to 8 and approaches the isoelectric point. In contrast, a reverse effect is observed in high salt conditions: the strength of attractive interactions decreases as the pH increases. The range of the pH effect can be related to ionization of histidine residues, particularly those located in the active site of the protein. The present observations point out the important role played by localized charges in crystallization conditions, whatever the precipitating agent.