Precision Analysis of Drug-Induced Conformational Changes of P-Glycoprotein Observed in AFM

Abstract

P-glycoprotein (Pgp) is an ATP-Binding Cassette transporter commonly overexpressed in cancer cells. This protein effluxes drugs out of the cell, leading to multi-drug resistance. Significant research interest surrounds the determination of the mechanism of action. A powerful single-molecule tool is the Atomic Force Microscope (AFM), which can directly image biomolecules in physiological buffer and temperature. Pgp reconstituted in supported lipid bilayers is imaged in the presence of cancer drugs and ATP-analogs to probe conformational changes. The high spatial resolution of the AFM must be matched by the precision of the post-imaging analysis. Previously, we have introduced the Hessian blob algorithm as a method of particle detection that minimizes user input [B.P. Marsh et al. Scientific Reports 8, 978 (2018)]. With the addition of scale-space Laplace interpolation to detect background levels, we increase the precision of particle heights, revealing previously unresolved populations of Pgp. Model selection techniques such as the Bayesian information criterion add to the objectivity of data interpretation. Together, these analyses refine our ability to deduce ligand-induced conformational shifts of Pgp in an unbiased manner.