Direct force measurement of single DNA–peptide interactions using atomic force microscopy

Chung, Jin; Shin, Dongjin; Kwak, June M.; Seog, Joonil

doi:10.1002/jmr.2269

Cited by 8 publications

(9 citation statements)

References 65 publications

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“…The binding strength of a selective interaction between dsDNA and an engineered peptide p007 was 42.1 pN (single-bond rupture event). The force-free off-rate ( k off ) was 0.32 ± 0.53 s −1 , the ( γ ) was 0.74 nm, and the mean lifetime ( t off ) of the complex was 3.1 s [24]. These values are comparable with the values obtained in other DNA-peptide or DNA-protein systems measured at the single-molecule level [25–28].…”

Section: Introductionsupporting

confidence: 72%

“…The binding probability and the rupture forces are then measured to characterize the interaction properties of the Ab and the antigen (DNA). It is noteworthy that, in addition to the optical trap, AFM-based rupture force spectroscopy has been widely used to study individual protein-ligand interactions [24–26, 28, 48]; however, the optical trap is more sensitive and accurate at lower forces of the order of piconewtons, while AFM usually operates in the nanonewton force range [49]. …”

Section: Discussionmentioning

confidence: 99%

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Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

et al. 2016

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Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10−3 s−1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies.

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Section: Introductionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

et al. 2016

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“…To provide forceÀextension behaviors of a single SELP molecule, we also stretched the SELP molecule normal to the mica surface, as described in our previous work. 67 Data Analysis. Height of the nucleus was determined from peak-to-peak distance in Gaussian fits of the time series data for the baseline and for the new nucleation site.…”

Section: Methodsmentioning

confidence: 99%

“…High resolution AFM imaging was performed using a silicon tip (MSNL, Bruker, CA) with a nominal tip radius of 2 nm. To provide force–extension behaviors of a single SELP molecule, we also stretched the SELP molecule normal to the mica surface, as described in our previous work …”

Section: Methodsmentioning

confidence: 99%

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

et al. 2013

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Self-assembly of amyloid nanofiber is associated with functional and pathological processes such as in neurodegenerative diseases. Despite intensive studies, stochastic nature of the process has made it difficult to elucidate molecular mechanisms for the key amyloid nucleation. Here, we investigated the amyloid nucleation of silk-elastinlike peptide (SELP) using time-lapse lateral force microscopy (LFM). By repeated scanning a single line on a SELP-coated mica surface, we observed sudden stepwise height increases, corresponds to nucleation of an amyloid fiber. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction, serves as the template for further selfassembly perpendicular to the scan direction. Such mechanically induced nucleation of amyloid fibrils allows positional and directional control of amyloid assembly in vitro, which we demonstrate by generating single nanofibers at predetermined nucleation sites.

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“…While the feasibility of measuring protein–carbohydrate interactions by AFM force spectroscopy has been demonstrated, important shortcomings remain to be addressed. To date, all relevant studies have utilized full-length proteins, and extending this approach to shorter bioactive peptides, as has been done for peptide–nucleic acid systems, , would greatly advance measurement capabilities, especially for proteins that are difficult to produce. Indeed, recombinant expression and purification of full-length HCV NS5A protein is technically challenging, and the N-terminal AH is often not included. , Hence, establishing measurement approaches based on synthetic bioactive peptides would be broadly useful and could provide a tractable means to examine NS5A AH BAAPP domain–phosphoinositide interactions.…”

mentioning

confidence: 99%

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

et al. 2017

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There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein–phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH–PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH–PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein–phosphoinositide interactions.

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Direct force measurement of single DNA–peptide interactions using atomic force microscopy

Cited by 8 publications

References 65 publications

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

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