The Mechanical Hierarchies of Fibronectin Observed with Single-molecule AFM

Oberhauser, Andrés F.; Badilla-Fernandez, Carmelu; Carrión‐Vázquez, Mariano; Fernández, Julio M.

doi:10.1016/s0022-2836(02)00306-6

Cited by 369 publications

(382 citation statements)

References 56 publications

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“…AFM studies on force-induced unfolding of FN III domains have shown that repeating distances of 28 nm between the rupture peaks corresponds to the fully unfolded length of a single FNIII domain (≈ 70 peptides x 0.4 nm = 28 nm ) 24 . If unfolding of the FNIII domain proceeds through an intermediate pathway, smaller peak spacings of 12 nm are observed 32 . We recently confirmed the characteristic unfolding of FNIII domains when using AFM to measure FN protein interactions on PPy doped with glycosaminoglycans 33 , thus a detailed analysis of the multiple rupture peaks was not repeated in this study.…”

Section: Resultsmentioning

confidence: 99%

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Gelmi

Higgins

Wallace

2013

Biochimica et Biophysica Acta (BBA) - General Subjects

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The interaction of ECM proteins is critical in determining the performance of materials used in biomedical applications such as tissue regeneration, implantable bionics and biosensing. Methods: To improve our understanding of ECM protein-conducting polymer interactions, we have used Atomic Force Microscopy (AFM) to elucidate the interactions of fibronectin (FN) on polypyrrole (PPy) doped with different glycosaminoglycans. Results: We were able to classify four main types of FN interactions, including those related to 1) non-specific adhesion, 2) protein unfolding and subsequent unbinding from the surface, 3) desorption and 4) interactions with no adhesion. FN adhesion on PPy/hyaluronic acid showed a significantly lower density of surface adhesion with the adhesion restricted to nodule structures, as opposed to their peripheries, of the polymer morphology. In contrast, PPy/chondroitin sulfate showed a significantly higher density of surface adhesion to the point where the distribution of adhesion effectively masked the topography. Through conductive AFM imaging, we found that the conductive regions correlated with regions of FN adhesion. Conclusions: Given that the conductivity requires doping of the polymer, these findings suggest that FN adhesion is mediated by interactions with chondroitin sulfate and hyaluronic acid at the polymer surface and may be indicative of specific interactions due to contributions from electrostatic attraction between the FN and sulfate/anionic groups of the dopants. General significance: This study demonstrates the ability of AFM to resolve the protein-conducting polymer interactions at the molecular and nanoscale level, which will be important for interfacing these polymer materials with biological systems. This article is part of a Special Issue entitled Organic Bioelectronics -Novel Applications in Biomedicine.

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Section: Resultsmentioning

confidence: 99%

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Gelmi

Higgins

Wallace

2013

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Furthermore, fully relaxed manually deposited Fn fibers do not contain a measurable population of Fn in the fully compact conformation (Fig. 3C and Smith et al, 2007 (Oberhauser et al, 2002). If three modules are unfolded mechanically, an extended Fn molecule which is estimated to be 130 nm in length prior unfolding (Engel et al, 1981), would thus increase in length to 206 nm (130+3 * (28.5-3.2)), or 1.6-fold.…”

Section: High Extensions Of Fn Fibers Involve Module Unfoldingmentioning

confidence: 97%

“…Major insights into force-induced unfolding pathways are available (Oberhauser et al, 2002;Samori et al, 2005;Wang et al, 2001), as well as high-resolution structural models relating unfolding of FnIII modules to altered protein functions as derived from steered molecular dynamics simulations (Craig et al, 2004a;Craig et al, 2004b;Craig et al, 2001;Vogel, 2006). Native, cell-derived Fn matrices, however, are not ideal systems for investigating forceinduced potentially altered structure-function relationships.…”

Section: Introductionmentioning

confidence: 99%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

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“…Unlike most modular proteins studied by atomic force microscopy, including titin 8,10-13 , tenascin 9 , fibronectin 14 , ubiquitin 15 , spectrin 16 and filamin 17 , whose elasticity typically follows a highly nonlinear entropic behaviour exemplified by the worm-like chain (WLC) model 18 ( Fig. 1c, dashed line), ankyrin is a hookean or linear spring for which the tension is proportional to the extension.…”

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confidence: 99%

Nanospring behaviour of ankyrin repeats

Lee

Abdi

Jiang

et al. 2006

Nature

352

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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The Mechanical Hierarchies of Fibronectin Observed with Single-molecule AFM

Cited by 369 publications

References 56 publications

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Nanospring behaviour of ankyrin repeats

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