title: A strategy for analyzing bond strength and interaction kinetics between Pleckstrin homology domains and PI(4,5)P2 phospholipids using force distance spectroscopy and surface plasmon resonance.
abstract: Phospholipids are important membrane components involved in diverse biological activities ranging from cell signaling to infection by viral particles. A thorough understanding of protein-phospholipid interaction dynamics is thus crucial for deciphering basic cellular processes as well as for targeted drug discovery. For any specific phospholipid-protein binding experiment, various groups have reported different binding constants, which are strongly dependent on applied conditions of interactions. Here, we report a method for accurate determination of the binding affinity and specificity between proteins and phospholipids using a model interaction between PLC-δ1/PH and phosphoinositide phospholipid PtdIns(4,5)P2. We developed an accurate Force Distance Spectroscopy (FDS)-based assay and have attempted to resolve the problem of variation in the observed binding constant by directly measuring the bond force. We confirm the FDS findings of a high bond strength of ∼0.19 ± 0.04 nN by Surface Plasmon Resonance (SPR) data analysis, segregating non-specific interactions, which show a significantly lower K(D) suggesting tight binding.
authors.affiliation: Stanford BioADD Laboratory, Stanford, California 94305, USA. jayraja@stanford.edu.
authors.forename: A V
authors.initials: AV
authors.lastname: Malkovskiy
authors.forename: D A
authors.initials: DA
authors.lastname: Wagh
authors.forename: F M
authors.initials: FM
authors.lastname: Longo
authors.forename: J
authors.initials: J
authors.lastname: Rajadas
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D001798:Blood Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47