Handheld chem/biosensor using extreme conformational changes in designed binding proteins to enhance surface plasmon resonance (SPR)

Lepak, Lori A.; Schnatz, Peter J.; Bendoym, Igor; Kosciolek, Derek J.; Koder, Ronald L.; Crouse, David T.

doi:10.1117/12.2222305

Cited by 3 publications

(4 citation statements)

References 16 publications

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“…Furthermore, engineered biosensors may substantially benefit from having a structural phase transition accompanying the binding of a target analyte. The changes in bulk material properties that result from the phase transition, such as the index of refraction in a monolayer of receptor proteins, offer the potential for large signal-to-noise gains over current biosensor technology, and of recreating naturally occurring molecular signaling mechanisms in an artificial context (23).…”

Section: Discussionmentioning

confidence: 99%

Designing heterotropically activated allosteric conformational switches using supercharging

Schnatz

Brisendine

Laing

et al. 2020

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

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Heterotropic allosteric activation of protein function, in which binding of one ligand thermodynamically activates the binding of another, different ligand or substrate, is a fundamental control mechanism in metabolism and as such has been a long-aspired capability in protein design. Here we show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into an allosteric ligand- and counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. As a result of the high surface-charge density, ligand binding to this protein is allosterically activated up to 1,300-fold by low concentrations of divalent cations and the polyamine spermine. To extend this process further using a natural protein, we similarly modified Escherichia coli cytochrome b562 and the resulting protein behaves in a like manner. These simple model systems not only establish a set of general engineering principles which can be used to convert natural and designed soluble proteins into allosteric molecular switches useful in biodesign, sensing, and synthetic biology, the behavior we have demonstrated––functional activation of supercharged intrinsically disordered proteins by low concentrations of multivalent ions––may be a control mechanism utilized by Nature which has yet to be appreciated.

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Section: Discussionmentioning

confidence: 99%

Designing heterotropically activated allosteric conformational switches using supercharging

Schnatz

Brisendine

Laing

et al. 2020

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

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“…The combination of the phase-changing supercharged DRNN(−27) chassis protein and computational design promises the ready development of sensitive, selective small-molecule sensors. While we describe a simple VX detector that can be built for less than $5000, this protein and the others designed using this method can be incorporated as the recognition element in any number of protein biosensor platforms that can take advantage of the large-scale phase change engendered by supercharging, including surface plasmon resonance-based protein-ligand complex formation sensors (31) and protein folding-based electrochemical detection schemes (32), to greatly enhance sensitivity.…”

Section: Vx Protein Selection Expression and Testingmentioning

confidence: 99%

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent

et al. 2022

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The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.

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“…Dramatically increasing the surface net charge of a well-folded protein, known as supercharging, can turn proteins into ligand-induced folders by destabilizing the folded state of the protein via charge–charge repulsion, and the additional folding energy that results from ligand binding is sufficient to drive the phase transition from an extended unliganded state to a compact folded bound state. Such modifications have been shown to improve protein solubility, stability, and reversibility while greatly increasing the electric field changes associated with ligand binding . For these reasons, supercharged IDPs represent an attractive target for biodevices; however, they have yet to be introduced into a sensing device.…”

Section: Introductionmentioning

confidence: 99%

“…Such modifications have been shown to improve protein solubility, stability, and reversibility 15 field changes associated with ligand binding. 16 For these reasons, supercharged IDPs represent an attractive target for biodevices; however, they have yet to be introduced into a sensing device. One of the main challenges is that IDP supercharging has only been used on designed and natural helical bundle proteins, which have simple folding topologies and are highly designable, 17 while the design rules to engineer this functionality into more complex folds, such as antibodies, have yet to be determined.…”

Section: ■ Introductionmentioning

confidence: 99%

Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments

Kim,

McCann,

Fortner

et al. 2024

J. Am. Chem. Soc.

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Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.

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Handheld chem/biosensor using extreme conformational changes in designed binding proteins to enhance surface plasmon resonance (SPR)

Cited by 3 publications

References 16 publications

Designing heterotropically activated allosteric conformational switches using supercharging

Designing heterotropically activated allosteric conformational switches using supercharging

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent

Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments

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