(405b) Single Molecule Force Spectroscopy for Nanoscience and Technology: What Can We Learn from Pulling on Single Macromolecules?

Zauscher, S., Duke University

Single molecule force spectroscopy (SMFS), performed with an atomic force microscope (AFM), allows for the direct study of inter- and intramolecular interactions in macromolecules and between macromolecules and surfaces. For example, SMFS has been used extensively over the past decade to better understand the conformational mechanics of biomacromolecules and the unbinding energetics of protein-ligand interactions. In SMFS, the deflection of a micro-cantilever is used to report the forces that are exerted upon stretching a molecule or ligand-receptor pair, tethered between the cantilever tip and a solid substrate. With the proliferation of AFMs over the last few years, SMFS has become available to a large number of researchers, and together with the growing ability to fabricate and manipulate nanoscale structures, SMFS has seen increasing use to answer technology and engineering driven questions. Here, we will discuss the principles of the technique, its strengths, limitations, and emerging opportunities and challenges.

To illustrate the power and limitations of the technique, we draw on recent work from our group where we used SMFS to study 1) the unbinding behavior of an HIV-1 Envelope (Env) glycoprotein (gp120), 2) the prolyl cis-trans isomerization in elastin-like polypeptides (ELP), and 3) the details of hydrophobic hydration of stimulus-responsive polyproteins.

The HIV-1 Env gp120 system is particularly interesting to the vaccine development community because gp120 is involved in initial docking to host T cells. When gp120 binds to CD4 on the host T cell, then a conformational change in gp120 follows that exposes a binding site for a chemokine receptor (CCR5 or CXCR4). In our experiments we measure the forces for gp120 unbinding from monoclonal antibodies (mAbs) A32 and 17b, which mimic the functions of host receptors CD4 and CCR5, respectively. SMFS allows a better understanding of the energy landscape associated with the unbinding event and thus provides a means to test the efficiency of potential reagents that inhibit this interaction.

We also used SMFS to investigate force-induced peptidyl-prolyl cis-trans isomerization. This transition is often fundamental for the biological activity of proteins, protein stability and folding pathways. In most of the previously reported experiments, cis-trans isomerization was catalyzed in a chymotrypsin-coupled, proline isomerase assay. Certain proteins, however, cannot be catalyzed using enzymes, and we show that force may provide an alternate trigger in these cases. We present evidence for this mechanism by Monte Carlo simulations of the force-extension curves using an elastically coupled two-state system. These results suggest that SMFS could be used to assay proline cis-trans isomerization in proteins and may thus have significant diagnostic utility.

Finally we present an approach we have developed that allows us to infer effects of changes in solvent quality and minor changes in molecular architecture on the molecular-elasticity of individual biomacromolecules. Specifically we show how changes in the effective Kuhn segment length can be used to interpret the hydrophobic hydration behavior of elastin-like polypeptides. Our results are intriguing as they suggest that SMFS can be used to study the subtleties of polypeptide-water interactions on the single molecule level.