Improved Single Molecule Force Spectroscopy Using Micromachined Cantilevers

Enhancing the short-term force precision of atomic force microscopy (AFM) while maintaining excellent long-term force stability would result in improved performance across multiple AFM modalities, including single molecule force spectroscopy (SMFS). SMFS is a powerful method to probe the nanometer-scale dynamics and energetics of biomolecules (DNA, RNA, and proteins). The folding and unfolding rates of such macromolecules are sensitive to sub-pN changes in force. Recently, we demonstrated sub-pN stability over a broad bandwidth (Δ<i>f</i> = 0.01–16 Hz) by removing the gold coating from a 100 μm long cantilever. However, this stability came at the cost of increased short-term force noise, decreased temporal response, and poor sensitivity. Here, we avoided these compromises while retaining excellent force stability by modifying a short (<i>L</i> = 40 μm) cantilever with a focused ion beam. Our process led to a ∼10-fold reduction in both a cantilever’s stiffness and its hydrodynamic drag near a surface. We also preserved the benefits of a highly reflective cantilever while mitigating gold-coating induced long-term drift. As a result, we extended AFM’s sub-pN bandwidth by a factor of ∼50 to span five decades of bandwidth (Δ<i>f</i> ≈ 0.01–1000 Hz). Measurements of mechanically stretching individual proteins showed improved force precision coupled with state-of-the-art force stability and no significant loss in temporal resolution compared to the stiffer, unmodified cantilever. Finally, these cantilevers were robust and were reused for SFMS over multiple days. Hence, we expect these responsive, yet stable, cantilevers to broadly benefit diverse AFM-based studies

Single-molecule force spectroscopy experiments unravel the nanomechanics of microbial adhesins.

<p>Series of force-distance profiles obtained by stretching adhesins from various microbial species (see text for details): single adhesion peaks reflecting specific recognition (A), sawtooth patterns with multiple force peaks corresponding to the force-induced unfolding of protein secondary structures (B), constant force plateaus originating from the mechanical unzipping of amyloid interactions formed between multiple adhesins (C), and single large adhesion force peaks with linear shapes obtained by pulling on Gram-positive bacterial pili (D). The arrows emphasize the characteristic force peaks in each case.</p

SpaCBA pili are glycosylated with mannose and fucose.

<p><b>(A) SpaCBA pili are glycosylated on <i>L</i>. <i>rhamnosus</i> GG cells—</b>Cell wall-associated proteins of <i>L</i>. <i>rhamnosus</i> GG wild type (1), the pilus-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357, 2) and the Δ<i>welE</i>::Tc^R mutant on which the pili are overexposed (CMPG5351, 3), were probed with mannose- and fucose-specific lectins (HHA and AAL resp.). Pili content was visualized by probing with SpaC antiserum (SpaC, black arrow and HMW: high molecular weight pili). Interference of the Msp1 glycoprotein was ruled out (open arrow). Blots and gels were performed in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(B) Purified pili are glycosylated—</b>SDS-PAGE separated pili (pool A) were stained with PAS glycostain and Sypro^® to visualize their protein content. Pili content was shown by probing of Western blotted samples with SpaC antiserum (HMW: high molecular weight pili). Purified Msp1 (open arrow) was used as a positive control. Representative gels are shown, experiment was carried out in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(C) SpaCBA pili bind mannose-specific lectins—</b>Purified pili fractions (PRM and pili pool B) were subjected to PAS glycostaining and both Sypro^® and Silver stain to visualize their protein content. Absence of 75 kDa signals on PAS and lectin blots rule out the interference Msp1 (cf. open arrow). Western blotted samples were probed with SpaC antiserum (HMW: high molecular weight pili) and the mannose-specific lectins HHA and GNA, visualizing the pili content of the samples and the presence of mannose, respectively. Representative gels and blots of in triplicate-repeated experiment. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(D&E) Mannose- and fucose-specific lectins bind SpaCBA pili—</b>Binding of lectins to plate-coated pili was measured to ELISA. Wells coated with coating buffer served as a negative control. Mannan and Lewis X were coated as a positive control for the mannose-specific HHA (<i>Hippeastrum</i> hybrid, D) and fucose-specific AAL (<i>Aleuria aurantia</i>, E) lectins, respectively. Error bars represent standard deviations of three independent experiments (paired t-test, p < 0.05)</p

Probing of fucose and mannose residues on <i>L</i>. <i>rhamnosus</i> GG pili using AFM.

<p>Fig 1A and 1C depict the adhesion forces and Fig 1B and 1D the rupture length histograms (n = 1024) obtained in buffer, from the interaction between <i>L</i>. <i>rhamnosus</i> GG wild type and fucose- and mannose-binding lectin probes (AAL and HHA resp.). In Fig 1E and 1F the force data for the interaction of a pili-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) with the two lectin probes are displayed. Insets show representative retraction force curves.</p

Immunogold labeling reveals colocalization of SpaA and fucose on SpaCBA pili.

<p>Immunoelectron microscopy double labeling of <i>L</i>. <i>rhamnosus</i> GG cells (A and B) and the Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) (C) with SpaA antiserum and the fucose-specific <i>Aleuria aurantia</i> lectin (AAL). Detection of SpaA and AAL was done using 5 nm (white arrows) and 10 nm gold particles (black arrows) respectively. The scale bar represents 500 nm. Original overall pictures are shown as insets of A and B.</p