Molecular Recognition Force Microscopy/SpectroscopyPeter Hinterdorfer
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz,Austria
E-mail: peter.hinterdorfer@jku.at
In molecular recognition force microscopy (MRFM), ligands are covalently attached toatomic force microscopy tips for the molecular recognition of their cognitive receptors onprobe surfaces. Using an appropriate tip surface chemistry protocol with 6-nm longheterobifunctional crosslinkers as key elements, the ligand density on the AFM tip issufficiently dilute for allowing single molecule studies. Our crosslinker library possesses
many different chemical endgroups for various functional coupling strategies. Interaction
forces between single receptor-ligand pairs are measured in force-distance cycles. A ligandcontaining tip is approached towards the receptors on the probe surface, which possibly leads
to formation of a receptor-ligand bond. The tip is subsequently retracted until the bond breaks
at a certain force (unbinding force). In force spectroscopy (FS), the dynamics of theexperiment is varied, which, in case of a single sharp activation barrier, reveals a logarithmic
dependence of the unbinding force on the force velocity. From this curve the barrier height
and width can be deduced, as shown on virus/cell receptor interactions. A more complex
energy landscape dominates the interaction of the nuclear import receptor importin  with thesmall GTPase Ran. The complex switches between two distinct conformational states ofdifferent binding strength. Our results support a model whereby functional control of Ranimportin is achieved by a population shift between pre-existing alternative conformation.
In another study it was shown that single molecule force measurements between
single-stranded DNA containing multiple methylcytosines and an anti-methylcytosine
antibody can survey the distances between methylcytosines with single nucleotide resolution.
Two step unbinding events in force curves corresponded to sequential dissociation of two
Fab-domains of one antibody from a single DNA molecule, with a distance in excellent
agreement with the contour length of nucleotides in between two methylcytosines. Using
different DNA sequences, the applicability for methylcytosine sequencing and the detectionof single nucleotide polymorphism at the single molecule level was demonstrated.
Finally, a method for the localization specific binding sites and epitopes with nm
positional accuracy by combining dynamic force microscopy with single molecule
recognition force spectroscopy is presented. A magnetically driven AFM tip containing aligand covalently bound via a tether molecule was oscillated at 5 nm amplitude while
scanning along the surface. Since the tether had a length of 6 nm, the ligand on the tip wasalways kept in close proximity to the surface and showed a high probabilty of binding when areceptor site was passed. Topography and recognition images were obtained simultaneously
using a specially designed electronic circuit. Maxima (Uup) and minima (Udown) of eachsinusoidal cantilever deflection period were depicted, with Udown driving the feedback loop torecord a height (topography) image and Uup providing the data for the recognition image. In
this way, topography and recognition image were gained simultaneously and independently
with nm lateral resolution. This method is capable of localizing distinct histones inchromation preparations and can visualize nm-sized receptor domains on cell surfaces.