A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc

Hunt, James; Keeble, Anthony H.; Dale, Robert E.; Corbett, Melissa K.; Beavil, Rebecca L.; Levitt, James A.; Swann, Marcus J.; Suhling, Klaus; Ameer-Beg, Simon; Sutton, Brian J.; Beavil, Andrew J.

doi:10.1074/jbc.m111.331967

Cited by 53 publications

(58 citation statements)

References 65 publications

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“…Dual labeling of IgE‐Fc with a FRET pair has previously provided valuable insight into the conformational changes associated with the binding of IgE to its high‐affinity receptor FcεRI, a key event in allergic disease because it results in the priming of mast cells with allergen‐specific IgE 14. An engineered IgE‐Fc construct (IgE‐Fc 2–4 ) containing N‐ and C‐terminally fused fluorescent proteins was used as a conformational FRET probe to monitor the IgE ⋅ FcεRI protein–protein interaction in solution 2c. Consistent with crystallographic data,15 increased FRET suggested a more bent conformational state of IgE when complexed with FcεRI.…”

Section: Methodsmentioning

confidence: 99%

“…Consistent with crystallographic data,15 increased FRET suggested a more bent conformational state of IgE when complexed with FcεRI. In contrast, binding of the anti‐IgE therapeutic IgG antibody omalizumab (Xolair) resulted in a reduced FRET signal, thus suggesting that the antibody‐bound IgE‐Fc adopts an inhibitory conformation that is unable to interact with FcεRI 2c. Subsequently, a similar construct, this time labeled through a mutated cysteine at position 289 and a BirA tag at the C terminus, enabled the selection and characterization of an antibody that captured IgE in a previously unobserved conformation 6c.…”

Section: Methodsmentioning

confidence: 99%

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Band Gap Engineering of BN Sheets by Interlayer Dihydrogen Bonding and Electric Field Control

et al. 2013

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An unconventional bonding: The presence of interlayer B-H⋅⋅⋅H-N dihydrogen bonds in hydrogenated bilayer BN nanosheets reduces the intrinsic large band gap of the chair-type BN conformation owing to interlayer charge transfer. In contrast, the insulating properties of the thermodynamically more stable stirrup-type bilayer BN can be engineered by applying an electric field. These results illuminate a new pathway for band gap engineering of 2D BN nanomaterials.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Band Gap Engineering of BN Sheets by Interlayer Dihydrogen Bonding and Electric Field Control

et al. 2013

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“…(6 and 8) cannot be used for large fluorophores, such as GFP, because the κ 2 value of 2/3, inherent in the R̄ 0 value of Eqs. (6) and (8), is not appropriate for a random isotropic population of FRET donors and acceptors that rotate slowly relative to the excited state lifetime of the donor [14,15,18,19]. In fact, under these conditions, formally called the static random isotropic orientational regime , there is no single value for κ 2 that is appropriate for all separations [15,16].…”

Section: Introductionmentioning

confidence: 99%

Estimating the distance separating fluorescent protein FRET pairs

Vogel

Meer

Blank

2014

Methods

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Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10 nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.

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“…[35] To visualize this change Hunt et al fused FP donor and acceptor groups to the N- (Cε2) and C-terminus (Cε4) of IgEFc. [36] FRET efficiency was substantial in the absence of receptor and increased significantly in its presence. The IgEFc biosensor is useful for investigating the molecular bases of antigen recognition and allergic response.…”

Section: Rigid-body Domain Movementmentioning

confidence: 99%

Protein Conformational Switches: From Nature to Design

Loh

2012

Chemistry A European J

147

131

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Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation—which can be carried out by a molecule as small as a thousand atoms or so—belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, bio-technology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and “smart” biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding–unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.

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A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc

Cited by 53 publications

References 65 publications

Band Gap Engineering of BN Sheets by Interlayer Dihydrogen Bonding and Electric Field Control

Band Gap Engineering of BN Sheets by Interlayer Dihydrogen Bonding and Electric Field Control

Estimating the distance separating fluorescent protein FRET pairs

Protein Conformational Switches: From Nature to Design

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