Molecular self-interactions of ribonuclease A revealed by isothermal titration calorimetry and self-interaction chromatography – Effects of anisotropy of protein surface charges

The roles of anisotropy of surface charge in the attraction (or repulsion) between molecules of same identity are probed by using myoglobin (Mb) and ribonuclease A (RNase A) as model proteins. To show the direction and extent of molecular interactions, we estimated second virial coefficients from the heat of protein dilution in isothermal titration calorimetry (ITC) experiments. While our ITC data of Mb were consistent with the study using popular self-interaction chromatography (SIC), those of RNase A in certain pH and salt settings contradicted. The results of size-exclusion chromatography supported the ITC findings. We propose that high anisotropy of surface charge of RNase A causes acute increase of induced attractive self-interaction at elevated ionic strength. Also, due to the use of surface-immobilized protein, the true in-solution behaviors of protein-protein interactions of proteins with high anisotropy of surface charge might not be revealed in SIC.