Carbon nanotube-based donor-acceptor devices are used in applications ranging from photovoltaics and sensors to environmental remediation. Non-covalent contacts between donor dyes and nanotubes are often used to optimize sensitization and scalability. However, inconsistency is often observed despite donor dye studies reporting strong donor-acceptor interactions. Here, we demonstrate that the dye binding location is an important factor in this process: we used coated-acceptor chromatic responses and find that dye binding is affected by the coating layer. The emission response to free- and protein-sequestered porphyrin was tested to compare direct and indirect dye contact. An acceptor complex that preferentially red-shifts in response to sequestered porphyrin was identified. We observe inconsistent optical signals that suggest porphyrin-dye interactions are best described as coating-centric; therefore, the coating interface must be considered in application and assay design.

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 b(562) 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.

Greatly increasing the magnitude of a protein's net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4 using simple molecular modeling, 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. Due to the high surface charge density, ligand binding to this protein is allosterically activated up to 1300-fold by low concentrations of divalent cations and the polyamine spermine. To extend this process further using a natural protein, we similarly modified E. coli cytochrome B562 and the resulting protein behaves in a like manner. These simple model systems allow us to derive and then experimentally validate a mass-action model for the coupled folding, binding and allosteric activation behavior of ligand-gated conformational switches, establishing 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. We further show that such supercharged proteins have a built-in 'dynamics dial', allowing us to manipulate the degree of disorder in the protein by changing the counterion concentration. This manipulation enables to examine in unprecedented detail the effect that dynamic disorder has on fundamental protein enzyme functions such as ligand binding and catalytic action.

Greatly increasing the magnitude of a protein's net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4 using simple molecular modeling to create a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction in biology. To demonstrate this process using more complex proteins, we then modified green fluorescent protein and the cytochrome B562, using a combination of simple modeling and electrostatic calculations to create proteins that unfold reversibly with decreasing ionic strength. These simple model systems allow us to derive and then experimentally validate a mass-action model for the coupled folding and binding behavior of ligand-gated conformational switches, establishing a set of engineering principles which can be used to convert natural and designed soluble proteins into molecular switches useful in biodesign and synthetic biology. We have applied these principles to a biosensing project in which supercharged IDPs are attached to nanostructured gold surfaces and conformational changes are sensed using surface plasmon resonance. An increase of the refractive index at the gold surface is caused by the ligand induced conformational change and can be detected via transmission surface plasmon resonance (SPR) spectroscopy. We calculate that the shift in the resonance wavelength of the surface plasmons is almost two orders of magnitude more than simple ligand binding to an already folded protein. Continuing SPR studies provide practical insight into the use of our model as conformational switches for biosensing devices.

We have designed a series of supercharged single-chain four-helix bundles as maquettes of intrinsically disordered proteins (IDPs). We show that a net charge per residue above ∼0.10e can impart enough electrostatic force to bias the folding equilibrium at low salt to an unfolded structure. We demonstrate that this behavior can be modulated as a function of pH and solution ionic strength, providing a wide functional dynamic range of folding energies. At the correct pH and salt concentration the proteins exhibit ligand-induced folding, and we have shown that this behavior can manifest as cooperative ligand binding. Furthermore, we are extending this supercharging to natural biopolymers, starting with green fluorescence protein, and we demonstrate that this behavior can be implanted on this fold. These adjustable characteristics have inspired a biosensing project in which we attach supercharged IDPs to a gold surface and sense conformational changes using surface plasmons. This conformational change will increase the refractive index at the gold surface, which will shift the angle of minimum reflectance. We calculate that the shift in the resonance angle caused by the ligand induced folding of an IDP is almost two orders of magnitude more than simple ligand binding to an already folded protein. Continuing reflectometry studies will provide practical insight into the use of our model as conformational switches for biosensing devices.

In this project we develop a handheld, portable, highly selective and sensitive chem/biosensor that has potential applications in both airborne and water-based environmental sensing. The device relies on a plasmonic chip of subwavelength-scale periodic gold rods engineered to resonate in the near infrared. The chip is functionalized with a novel class of proteins that exhibit large conformational changes upon binding to a specific target analyte. The subsequent change in local refractive index near the surface of the gold is one to two orders of magnitude greater than current conventional methods, which produces a readily measurable 5 to 10 percent difference in light transmission. This allows us to forgo traditional, bulky tabletop setups in favor of a compact form factor. Using commercially available optics to construct a transmission-based optical train, measured changes in bulk refractive index are presented here. While synthesis of binding protein efforts are focused on heme as analyte for proof of concept validation, the functionalized protein can be engineered to pair with a wide variety of analytes with minimal alterations to the plasmonic chip or device design. Such flexibility allows for this device to potentially meet the needs of first responders and health care professionals in a multitude of scenarios.

We present research results centered on development of a highly sensitive handheld chem/biosensor device using a novel class of engineered proteins, designed to undergo extreme conformational changes upon binding their target, which in turn cause extreme changes in refractive index in the protein layer. These proteins are attached to a detector chip with a structured metasurface, to translate the refractive index change into an enhanced shift in surface plasmon resonances (SPR), thereby improving the sensitivity of the overall detector relatively to current commercially available SPR systems. Theoretical calculations have demonstrated the potential of the conformational changes in the engineered proteins to provide the desired change in refractive index. A plasmonic chip with a simple grating metasurface structure was designed to maximize the SPR shift. A prototype chip and a prototype for the overall device housing were fabricated with the inclusion of all other required (commercially available) optical components. The proposed device holds considerable promise as a low-cost, highly sensitive, field-deployable detection system for chemical and biological toxins.

We present research results centered on development of a highly sensitive handheld chem/biosensor device using a novel class of engineered proteins, designed to undergo extreme conformational changes upon binding their target, which in turn cause extreme changes in refractive index in the protein layer. These proteins are attached to a detector chip with a structured metasurface, to translate the refractive index change into an enhanced shift in surface plasmon resonances (SPR), thereby improving the sensitivity of the overall detector relatively to current commercially available SPR systems. Theoretical calculations have demonstrated the potential of the conformational changes in the engineered proteins to provide the desired change in refractive index. A plasmonic chip with a simple grating metasurface structure was designed to maximize the SPR shift. A prototype chip and a prototype for the overall device housing were fabricated with the inclusion of all other required (commercially available) optical components. The proposed device holds considerable promise as a low-cost, highly sensitive, field-deployable detection system for chemical and biological toxins.