Binding assay for low molecular weight analytes based on reflectometry of absorbing molecules in porous substrates

Stephan, Milena; Kramer, Corinna; Steinem, Claudia; Janshoff, Andreas

doi:10.1039/c4an00009a

Cited by 8 publications

(9 citation statements)

References 28 publications

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“…The optical displacement difference in nanometers between both channels allowed for the establishment of a binding curve followed by a dissociation curve. The SKI Pro software algorithm was used to calculate K on , K off and K obs values, from which a K d value was estimated (Latterich & Corbeil, 2008; Sanchez et al , 2010; Stephan et al , 2014).…”

Section: Methodsmentioning

confidence: 99%

Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants

Retamal

Abed

Corbeil

et al. 2014

Journal of General Virology

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Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Qué bec City, QC, Canada mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants, which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here, we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E, N173D and K256E) and eight novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), three (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I and K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in three different Bris07 escape mutants, appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.

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Section: Methodsmentioning

confidence: 99%

Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants

Retamal

Abed

Corbeil

et al. 2014

Journal of General Virology

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“…RIfS is an optical technique that allows label-free investigations of protein binding to a specific receptor lipid embedded in SLBs (27,(31)(32)(33)(34)(35). The adsorption process is detected as an increase in optical thickness, OT ¼ n d, i.e., the product of the refractive index n and the physical layer thickness d. RIfS experiments were employed to analyze whether ezrin wt or ezrin T567D adsorbed to either the receptor lipid DOGS-Ni-NTA or PIP 2 results in a difference in OT.…”

Section: Analysis Of Ezrin-lipid Interactions By Rifsmentioning

confidence: 99%

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Shabardina

Kramer

Gerdes

et al. 2016

Biophysical Journal

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Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

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“…Reflectometric Interference Spectroscopy-Reflectometric interference spectroscopy measurements were performed using a custom-built setup equipped with a NanoCalc-2000 spectrometer (OceanOptics Germany GmbH, Ostfildern, Germany) (60). SiO 2 wafers with an oxide layer of 5 m were incubated in a solution of NH 3 and H 2 O 2 (H 2 O/NH 3 /H 2 O 2 , 5:1:1) for 20 min at 70°C.…”

Section: Methodsmentioning

confidence: 99%

“…After rinsing with buffer, the resulting membrane was treated with a bovine serum albumin solution (1 mg/ml) to prevent nonspecific protein adsorption. After again rinsing with buffer, ENTH was added, and spectra were recorded every 2 s and the optical thickness determined (60).…”

Section: Methodsmentioning

confidence: 99%

Epsin N-terminal Homology Domain (ENTH) Activity as a Function of Membrane Tension

Gleisner

Kroppen

Fricke

et al. 2016

Journal of Biological Chemistry

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The epsin N-terminal homology domain (ENTH) is a major player in clathrin-mediated endocytosis. To investigate the influence of initial membrane tension on ENTH binding and activity, we established a bilayer system based on adhered giant unilamellar vesicles (GUVs) to be able to control and adjust the membrane tension covering a broad regime. The shape of each individual adhered GUV as well as its adhesion area was monitored by spinning disc confocal laser microscopy. Control of in a range of 0.08-1.02 mN/m was achieved by altering the Mg 2؉ concentration in solution, which changes the surface adhesion energy per unit area of the GUVs. Specific binding of ENTH to phosphatidylinositol 4,5-bisphosphate leads to a substantial increase in adhesion area of the sessile GUV. At low tension (<0.1 mN/m) binding of ENTH can induce tubular structures, whereas at higher membrane tension the ENTH interaction deflates the sessile GUV and thereby increases the adhesion area. The increase in adhesion area is mainly attributed to a decrease in the area compressibility modulus K A . We propose that the insertion of the ENTH helix-0 into the membrane is largely responsible for the observed decrease in K A , which is supported by the observation that the mutant ENTH L6E shows a reduced increase in adhesion area. These results demonstrate that even in the absence of tubule formation, the area compressibility modulus and, as such, the bending rigidity of the membrane is considerably reduced upon ENTH binding. This renders membrane bending and tubule formation energetically less costly.Clathrin-mediated endocytosis is one of the key metabolic pathways for the uptake of macromolecules into eukaryotic cells (1-4). Driven by a chain of remodeling events and an elaborate set of proteins acting in an orchestrated manner, an almost flat patch of plasma membrane is transformed into a closed, cargo-containing vesicle. As plasma membrane shape transformation is associated with significant local bending of the membrane, the process is highly sensitive to lateral membrane tension. Plasma membrane tension originates from two primary sources; that is, hydrostatic pressure across the lipid bilayer and cytoskeleton-membrane adhesion (5). Depending on the cell type, plasma membrane tensions span a range of roughly 0.003-0.45 mN/m (5-8). Even though it had become clear already in the late 1990s that tension plays a role in exoand endocytosis (9, 10), only in recent years has significant evidence been accumulated that membrane tension is of utmost importance for processes that rely on membrane remodeling (11-14). Cells actively maintain and regulate their membrane tension and use it to control exo-and endocytosis (15). In K562 cells it has been reported that endocytosis is completely suppressed under hypoosmotic conditions (16). Generally, high lateral membrane tension suppresses membrane deformation as both stretching the lipid bilayer and opening bonds between the cytoskeleton and the plasma membrane requires a large amount of energy (17).One protein...

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Binding assay for low molecular weight analytes based on reflectometry of absorbing molecules in porous substrates

Cited by 8 publications

References 28 publications

Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants

Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Epsin N-terminal Homology Domain (ENTH) Activity as a Function of Membrane Tension

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