Sensing specific molecular interactions with the atomic force microscope

Florin, Ernst‐Ludwig; Rief, Matthias; Lehmann, Horst; Ludwig, Markus; Dornmair, C.; Moy, Vincent T.; Gaub, Hermann E.

doi:10.1016/0956-5663(95)99227-c

Cited by 452 publications

(361 citation statements)

References 25 publications

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“…1A). The spring constant of each individual AFM cantilever (Si 3 N 4 tip, TwinTips, Digital Instruments, Santa Barbara, CA, or microlevers from Thermomicroscopes, Sunnyvale, CA) was calibrated in solution, using the equipartition theorem (24). The step time response of our force-clamp͞ramp system was Ϸ20 ms and the uncertainty of the force magnitude during the ramp, because of the cantilever drift, was Ͻ50 pN (16).…”

Section: Methodsmentioning

confidence: 99%

Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM

Marszałek

Oberhauser

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

136

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Under a stretching force, the sugar ring of polysaccharide molecules switches from the chair to the boat-like or inverted chair conformation. This conformational change can be observed by stretching single polysaccharide molecules with an atomic force microscope. In those early experiments, the molecules were stretched at a constant rate while the resulting force changed over wide ranges. However, because the rings undergo force-dependent transitions, an experimental arrangement where the force is the free variable introduces an undesirable level of complexity in the results. Here we demonstrate the use of force-ramp atomic force microscopy to capture the conformational changes in single polysaccharide molecules. Force-ramp atomic force microscopy readily captures the ring transitions under conditions where the entropic elasticity of the molecule is separated from its conformational transitions, enabling a quantitative analysis of the data with a simple two-state model. This analysis directly provides the physico-chemical characteristics of the ring transitions such as the width of the energy barrier, the relative energy of the conformers, and their enthalpic elasticity. Our experiments enhance the ability of single-molecule force spectroscopy to make high-resolution measurements of the conformations of single polysaccharide molecules under a stretching force, making an important addition to polysaccharide spectroscopy. S ingle molecule force spectroscopy has become an important tool to examine the conformations of proteins and polysaccharide molecules under a stretching force (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Recent experiments have demonstrated that a force field can trigger conformational changes in these molecules that cannot be observed by traditional NMR or x-ray crystallographic techniques. Force spectroscopy involves measuring the force required to extend a molecule by a certain amount (2-10). The data are compiled into a forceextension relationship that gives a characteristic fingerprint for the conformational transitions of the molecule under study. For example, force-extension relationships of polysaccharides such as amylose and dextran (13, 14) display a characteristic plateau (2, 4) that marks forced transitions of the glucose monomers from their chair conformation to the boat-like conformation (4). Similarly, two distinct plateaus in the force-extension curve of pectin (13, 14) report a transition of the galactose rings from the chair conformation to the boat conformation and a subsequent flip of the boat to the inverted chair conformation (5). These observations provide an interesting perspective on the behavior of polysaccharides under mechanical tension (15) and suggest that fingerprints of forced conformational transitions can be used as a means for identifying single polysaccharides molecules in solution (9).In the most typical atomic force microscopy (AFM) experiment, a single molecule adsorbed between a substrate and the cantilever tip is extended vertically at a constant rate while t...

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Section: Methodsmentioning

confidence: 99%

Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM

Marszałek

Oberhauser

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

136

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“…The spring constant of each MLCT-AUHW micro-cantilever (Veeco) was calibrated by using the energy equipartition theorem as described in ref. 28. Molecules were picked up for stretching measurements by gently touching the substrate with the AFM tip, exploiting a nonspecific adsorption of ankyrin to the tip.…”

Section: Methodsmentioning

confidence: 99%

Nanospring behaviour of ankyrin repeats

Lee

Abdi

Jiang

et al. 2006

Nature

351

300

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Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla 1 . Ankyrin repeats contain antiparallel a-helices that can stack to form a superhelical spiral 2 . Visual inspection of the extrapolated structure of 24 ankyrin-R repeats 2 indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles 3-5 . Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common aminoacid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.The atomic structure of 12 ankyrin-R repeats suggests that ankyrin stacks composed of n $ 24 repeats should form a full superhelical turn with putative spring properties 2,3 . We used an atomic force microscope (AFM) to identify individual stacks of 24 ankyrin-B repeats ( Supplementary Fig. S2) and found that they do indeed have a hook-like shape 2 with the molecules' end-to-end distance closely matching the ,12 nm determined for the extrapolated structure 2 (Fig. 1a). Thus, the AFM images strongly suggest that the engineered protein, bearing at its terminus a glutathione S-transferase (GST) module, is correctly folded and does not aggregate. These conclusions are further supported by circular dichroism and hydrodynamic measurements (Supplementary Table 1 and Supplementary Fig. S1).For elasticity measurements, heptahistidine-tagged polypeptides containing 24 ankyrin-B repeats with or without GST, or 12 repeats with GST, were immobilized on a glass surface bearing the metal chelate N-nitrilotriacetic acid (NTA) 6,7 (Fig. 1a). Molecules were stretched vertically, in solution, by the AFM cantilever, and their length and tension were measured with subnanometre and ,10 pN precision [8][9][10] . Most trials revealed complex force-extension profiles with irregularly spaced force peaks typical of multiple molecules ( Supplementary Fig. S4a). However, ,5% of the force-extension curves had simple and consistent features that, we argue, represent LETTERSFigure 1 | Atomic force microscopy measurements reveal the linear elasticity of ankyrin-B repeats. a, The extrapolated structure of 24 ankyrin-R repeats 2 and a diagram of the elasticity measurement on a His-tagged ankyrin fragment bound to NTA (red handles) and stretched with the AFM cantilever. b-e, Force-extension curves of individual ankyrins: 24 repeats with GST (b-d); 24 repeats with no GST (e)....

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“…All single-molecule force measurements were performed on a custom-built atomic force microscope. Calibration of cantilevers was done in solution by using the equipartition theorem (15,16). This method provides a resolution in force of Ϸ10%.…”

Section: Methodsmentioning

confidence: 99%