Force spectroscopy of the double-tethered concanavalin-A mannose bond.
ABSTRACT We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.
Article: Molecule-level imaging of Pax6 mRNA distribution in mouse embryonic neocortex by molecular interaction force microscopy.[show abstract] [hide abstract]
ABSTRACT: Detection of the cellular and tissue distributions of RNA species is critical in our understanding of the regulatory mechanisms underlying cellular and tissue differentiation. Here, we show that an atomic force microscope tip modified with 27-acid dendron, a cone shaped molecule with 27 monomeric units forming its base, can be successfully used to map the spatial distribution of mouse Pax6 mRNA on sectioned tissues of the mouse embryonic neocortex. Scanning of the sectioned tissue with a 30-mer DNA probe attached to the apex of the dendron resulted in detection of the target mRNA on the tissue section, permitting mapping of the mRNA distribution at nanometer resolution. The unprecedented sensitivity and resolution of this process should be applicable to identification of molecular level distribution of various RNAs in a cell.Nucleic Acids Research 12/2008; 37(2):e10. · 8.03 Impact Factor
Article: Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.[show abstract] [hide abstract]
ABSTRACT: p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.International Journal of Nanomedicine 01/2011; 6:3011-9. · 3.13 Impact Factor