Nanopore Analysis on the Drug-Induced Conformation Change of a p53-Linker-Mouse Double Minute 2 Protein Complex

Detection of conformational changes in proteins by protein-protein interaction (PPI) is a key issue in developing drug screening platforms. In order to effectively investigate the conformational change in a protein at a single-molecule level, we propose the use of nanopore detection to identify protein conformational changes resulting from protein-protein interactions and their inhibition by Nutlin-3. We designed a protein complex comprising a p53 peptide and a mouse double minute 2 (MDM2) linked by 6 amino acids, transforming its shape from globular to dumbbell structure by inhibition of interaction between p53 peptide and MDM2. In the NMR experiment, no distinguished crosspeaks were observed upon Nutlin-3 addition. However, the nanopore experiment clearly showed double-peak signals with the addition of Nutlin-3. The observed fraction of the double-peak among single-peak signals increased from 8.77% to 22.03% with a concurrent increase in the Nutlin-3 concentration from a molar ratio of 1 to 10-fold. From the nanopore data, we estimated the dwell time for the elongated form of Nutlin-3-bound protein, which traverses for a longer duration (∼2 times) than the globular form. Finally, the hydrodynamic diameter of the local peak of the double-peak signal was calculated and compared with the X-ray crystallography results. This approach shows feasibility of the nanopore detection to verify the protein conformational change by inhibition of protein-protein interaction at a single-molecule level.