Protein thermal stability and phospholipid-protein interaction investigated by capillary isoelectric focusing with whole column imaging detection.
CIEF with whole column imaging detection (WCID) is an attractive technique for studying protein reaction and protein-ligand interaction due to its fast separation, simple operation, and high efficiency. In this study, two interesting applications by the CIEF-WCID were developed, involving the study of protein thermal stability and phospholipid-protein interaction. Four proteins (beta-lactoglobulin B, trypsin inhibitor, phosphorylase b, and trypsinogen) with different pI, and two types of phospholipids, including phosphatidylcholine (PC) and phosphatidylserine (PS), were used for this purpose. First, the altered CIEF profiles of four proteins were exhibited due to conformational changes resulting from protein denaturation induced by a high incubation temperature at 60 degrees C. It was demonstrated that the addition of a zwitterionic phospholipid (PC) played a crucial role in the thermal stability of targeted proteins, especially for trypsin inhibitor whose thermal stability was promoted with the addition of the PC vesicles at 60 degrees C. Second, the zwitterionic (PC) and acidic (PS) phospholipids displayed completely different interactions with the proteins. The addition of PS vesicles modified the zwitterionic phospholipids to carry negative charges, which correspondingly changed the interaction between the phospholipid and the protein. Our study demonstrates that the CIEF-WCID is a powerful approach to study protein reaction and protein-ligand interaction with high efficiency, high selectivity, and fast separation.
Bo
Tao
T
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.
Pawliszyn
Janusz
J
D002845:Chromatography / Q000379:methods*; D006358:Hot Temperature; D007525:Isoelectric Focusing / Q000379:methods*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D010743:Phospholipids; D011506:Proteins