Single-molecule scale quantification reveals interactions underlying protein–protein interface: from forces to non-covalent bonds

Abstract

Using atomic force microscopy-based single-molecule force spectroscopy to quantify noncovalent binding between BAX and Bcl-2, and observing that complicated multivalent binding interactions induced stable BAX/Bcl-2 complexes.

Molecular-Scale Investigations Reveal the Effect of Natural Polyphenols on BAX/Bcl-2 Interactions

“…In 24.2% of the total curves, specific unbinding events of BAX/Bcl-2 interaction pairs are observed ( Figure 1 D). In our previous studies, we demonstrated the existence of a specific interaction between BAX and Bcl-2 using different system configurations (blocking and control experiments) [ 23 ]. After a gradually increase in the polyphenol injection concentration (ranging from 10 µM to 50 µM), we observed a progressive decrease in BP with polyphenol concentration ( Figure 1 D).…”

“…We assumed that the forces follow a mixed Gaussian distribution with multiple peaks corresponding to multivalent binding between protein pairs ( Figure S3 ). Multivalent binding between BAX/Bcl-2 interfaces has been previously demonstrated [ 23 ]. We observed that the force distributions all exhibited periodic peaks.…”

“…The binding kinetics between BAX/Bcl-2 are mainly mediated by hydrogen bonds, hydrophobic interactions, van der Waals forces, and electrostatic interactions [ 23 ]. Polyphenol binding to Bcl-2 alters the noncovalent interactions of PPIs, and multiple driving forces reduce the stability of the BAX/Bcl-2 complex.…”

“…AFM tips and substrates were prepared as previously described [ 23 , 47 ]. Briefly, Au-coated tips (ContGB-G with tip radius < 25 nm, an apex half-cone angle of 10° and resonance frequency of 13 kHz, and HQ: CSC38 with tip radius < 35 nm, full cone angle of 40° and resonance frequency of 10 kHz) and substrates were rinsed with acetone, ethanol, piranha solution (H 2 SO 4 :H 2 O 2 = 7:3, v/v ), and ultrapure water to remove contaminants.…”

“…In the field of drug discovery, it also allows the evaluation of protein–drug interactions or drug-modulating PPIs under physiological or pathological conditions [ 21 , 22 ], providing valuable information on drug action. Previously, we reported the use of AFM to reveal the interaction force between BAX and Bcl-2 [ 23 ]. Through single-molecule force spectroscopy (SMFS), we revealed the existence of complex non-covalent interactions at the BAX/Bcl-2 interface.…”