Recognition Force Spectroscopy Studies of the NTA-His6 Bond

Kienberger, Ferry; Kada, Gerald; Gruber, Hermann J.; Pastushenko, V. F.; Riener, Christian K.; Trieb, Maria; Knaus, Hans-Günter; Schindler, Hansgeorg; Hinterdorfer, Peter

doi:10.1002/(sici)1438-5171(200004)1:1<59::aid-simo59>3.0.co;2-v

Cited by 114 publications

(93 citation statements)

References 32 publications

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“…Measurement of the force required to rupture the metalhistidine complex has been accomplished by several groups, although not at the loading rate used here (36)(37)(38)(39). Iron(III) complexes require a significantly larger rupture force than those based on Ni 2ϩ .…”

Section: Resultsmentioning

confidence: 99%

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

Snyder

Lee

Marszałek

et al. 2007

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

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A cantilever device based on competitive binding of an immobilized receptor to immobilized and soluble ligand and capable of measuring solution-phase thermodynamic quantities is described. Through multiple binary queries, the device stochastically measures the probability of the formation of a bound complex between immobilized protein and immobilized ligand as a function of soluble ligand concentration. The resulting binding isotherm is described by a binding polynomial consisting of the activities of soluble and immobilized ligand and binding constants for the association of immobilized protein with free and immobilized ligand. Evaluation of the polynomial reveals an association constant for the formation of a complex between immobilized ligand and immobilized protein close to that for the formation of complex between soluble protein and soluble ligand. The methodology lays the foundation for construction of practical portable sensing devices.force spectroscopy ͉ ligand binding ͉ sensors ͉ competitive binding T echnologies that permit the rapid quantitative determination of analytes have myriad applications in point-of-care diagnostics and environmental and food safety monitoring. Most rely on the formation of specific complexes between an analyte and a receptor and require an experimentally observable measure of binding. By far, the most commonly used signal is a change in optical properties, either absorptive or emissive. However, many systems of interest lack useable optical properties, either because of the inherent optical properties of the analytes themselves or because of the characteristics of the sample milieu, and in recent years a number of general approaches have emerged for the measurement of intermolecular binding. During the past several years, titration microcalorimeters have become commercially available (1, 2). These instruments measure the heat evolved during ligand binding as a function of ligand concentration; this information can be fit to a simple binding isotherm to reveal binding free energies and enthalpies. Another group of techniques, typified by surface plasmon resonance and quartz crystal microbalance, use changes in the electro-optical properties of a surface as a small-or macromolecular species is deposited (3-5). This signal is used to determine kinetic values for binding and unbinding, and the ratio of these parameters furnishes a binding constant. Although broadly applicable, both techniques require long sampling times (hours) and have material and operational characteristics that preclude their utility in field applications.Dynamic force spectroscopy, typified by the atomic force microscope (AFM) and the optical trap, measures the force required to unbind individual molecular complexes (6-20). Unbinding forces are not simply related to free energies or to association rates, because the application of force necessarily removes the bound complex from an equilibrium condition (21). The study of unbinding kinetics by dynamic force spectroscopy is further complicated by the applic...

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Section: Resultsmentioning

confidence: 99%

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

Snyder

Lee

Marszałek

et al. 2007

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

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“…This yields a force of % 0.2 pN on each peptide molecule, and since they are embedded in a 10 mm thick NTA-copolymer matrix, the actual force on the single hydrogen bond maintaining the peptide structure is substantially lower. In relation to the fact that the average unfolding force of protein secondary structures and the unbinding force to dissociate a NTA-His6 bond are in the range of 100-200 pN, [36,37] we can safely exclude this possibility.…”

mentioning

confidence: 99%

A Novel ATR‐FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species

et al. 2007

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We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR‐FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC™) chips containing about 10 μm thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR‐FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)‐tagged synthetic peptides of 25 (25‐His6) and 48 (48‐His6) amino acids to the NTA‐groups and 3) attachment of a ligand, mesyl amide, to the surface‐bound 48‐His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of ≈100 mg mL−1 and a dissociation constant of 116±11 μM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR‐FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm−1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand‐alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set‐ups.

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“…Figure 1D shows a representative approach-retract cycle for the described experimental setup. To avoid disruption of the NTA-His6 bond (Kienberger et al 2000a) and damage of the immobilized proteins, the contact loading force was limited to values between 50-300 pN. The unbinding force f u was measured from the maximum deflection signal prior to pulloff on the retraction curve; in the case of multiple-event retraces, only the last pulloff jump was considered.…”

Section: Armadillo B-catenin Forms Two Complexes With Tcf4mentioning

confidence: 97%

Molecular plasticity of β‐catenin: New insights from single‐molecule measurements and MD simulation

2007

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The multifunctional protein, b-catenin, has essential roles in cell adhesion and, through the Wnt signaling pathway, in controlling cell differentiation, development, and generation of cancer. Could distinct molecular forms of b-catenin underlie these two functions? Our single-molecule force spectroscopy of armadillo b-catenin, with molecular dynamics (MD) simulation, suggests a model in which the cell generates various forms of b-catenin, in equilibrium. We find b-catenin and the transcriptional factor Tcf4 form two complexes with different affinities. Specific cellular response is achieved by the ligand binding to a particular matching preexisting conformer. Our MD simulation indicates that complexes derive from two conformers of the core region of the protein, whose preexisting molecular forms could arise from small variations in flexible regions of the b-catenin main binding site. This mechanism for the generation of the various forms offers a route to tailoring future therapeutic strategies.

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Recognition Force Spectroscopy Studies of the NTA-His6 Bond

Cited by 114 publications

References 32 publications

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

A Novel ATR‐FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species

Molecular plasticity of β‐catenin: New insights from single‐molecule measurements and MD simulation

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