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)....

Recent atomic force microscopy stretching measurements of single polysaccharide molecules suggest that their elasticity is governed by force-induced conformational transitions of the pyranose ring. However, the mechanism of these transitions and the mechanics of the pyranose ring are not fully understood. Here we use steered molecular dynamics simulations of the stretching process to unravel the mechanism of forced conformational transitions in 1,6 linked polysaccharides. In contrast to most sugars, 1,6 linked polysaccharides have an extra bond in their inter-residue linkage, C5-C6, around which restricted rotations occur and this additional degree of freedom increases the mechanical complexity of these polymers. By comparing the computational results with the atomic force microscopy data we determine that forced rotations around the C5-C6 bond have a significant and different impact on the elasticity of alpha- and beta-linked polysaccharides. Beta-linkages of a polysaccharide pustulan force the rotation around the C5-C6 bonds and produce a Hookean-like elasticity but do not affect the conformation of the pyranose rings. However, alpha-linkages of dextran induce compound conformational transitions that include simultaneous rotations around the C5-C6 bonds and chair-boat transitions of the pyranose rings. These previously not-recognized transitions are responsible for the characteristic plateau in the force-extension relationship of dextran.

Rrp44 (Dis3) is a key catalytic subunit of the yeast exosome complex and can processively digest structured RNA one nucleotide at a time in the 3’ to 5’ direction. Its motor function is powered by the energy released from the hydrolytic nuclease reaction instead of ATP hydrolysis as in conventional helicases. Single-molecule fluorescence analysis revealed that instead of unwinding RNA in single base pair steps, Rrp44 accumulates the energy released by multiple single nucleotide step hydrolysis-reactions until about four base pairs are unwound in a burst. Kinetic analyses showed that RNA unwinding, not cleavage or strand release, determines the overall RNA degradation rate, and that the unwinding step size is determined by the nonlinear elasticity of the Rrp44/RNA complex, but not by duplex stability.

λ exonuclease degrades one strand of duplex DNA in the 5’-3’ direction to generate a 3’ overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure and most studies have focused on the processive phase. Here, we use single molecule FRET to reveal three phases of λ exonuclease reactions: initiation, distributive and processive phases. The distributive phase occurs at early reactions where the 3’ overhang is too short for a stable engagement with the enzyme. A mismatched base is digested five times slower than a Watson-Crick paired base and concatenating multiple mismatches has a cooperatively negative effect, highlighting the crucial role of basepairing in aligning the 5’ end toward the active site. The rate-limiting step during processive degradation appears to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.

Self-assembled polymers whose main chains are defined by reversible DNA base pairing form bridges between the tip of an atomic force microscope and substrate. The forces associated with the rupture of these assemblies are independent of polymer bridge length, and they resemble those expected for the isolated associations defining the polymer bridges. The assembly is reversible and is inhibited by a competitive, nonpolymerizing oligonucleotide. Noncomplementary polymer brush layers do not bridge, and therefore, the forces result from specific molecular recognition events. The length distribution of the bridges differs greatly from that of the polymers in solution, and thus the bridging is responsive to the spatial constraints of the environment.

The elasticity of single amylose chains in solvents that promote or inhibit the formation of inter‐residue hydrogen bonds was examined by AFM (see picture). Hydrogen bonds were found to rigidify the amylose chain in solvents of low dielectric constant, and the strength of inter‐residue hydrogen bonds in sugars can be measured.

Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, beta-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed alpha-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10-15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs.

Recent single-molecule atomic force microscopy (AFM) experiments have revealed that some polysaccharides display large deviations from force-extension relationships of other polymers which typically behave as simple entropic springs. However, the mechanism of these deviations has not been fully elucidated. Here we report the use of novel quantum mechanical methodologies, the divide-and-conquer linear scaling approach and the self-consistent charge density functional-based tight binding (SCC-DFTB) method, to unravel the mechanism of the extensibility of the polysaccharide amylose, which in water displays particularly large deviations from the simple entropic elasticity. We studied the deformations of maltose, a building block of amylose, both in a vacuum and in solution. To simulate the deformations in solution, the TIP3P molecular mechanical model is used to model the solvent water, and the SCC-DFTB method is used to model the solute. The interactions between the solute and water are treated by the combined quantum mechanical and molecular mechanical approach. We find that water significantly affects the mechanical properties of maltose. Furthermore, we performed two nanosecond-scale steered molecular dynamics simulations for single amylose chains composed of 10 glucopyranose rings in solution. Our SCC-DFTB/MM simulations reproduce the experimentally measured force-extension curve, and we find that the force-induced chair-to-boat transitions of glucopyranose rings are responsible for the characteristic plateau in the force-extension curve of amylose. In addition, we performed single-molecule AFM measurements on carboxymethyl amylose, and we found that, in contrast to the results of an earlier work by others, these side groups do not significantly affect amylose elasticity. By combining our experimental and modeling results, we conclude that the nonentropic elastic behavior of amylose is governed by the mechanics of pyranose rings themselves and their force-induced conformational transitions.