Biology subjects

Schrunk, E.

Publications and source records attributed to Schrunk, E..

3 recordsLinked to original sources

bioRxiv2025

A modular method for rapidly prototyping targeted gas vesicle protein nanoparticles

Gas vesicles (GVs) are air-filled protein nanoparticles which are proving to be useful in a number of biomedical applications. We hypothesized that it could be possible to develop a modular method for creating rapidly prototyped GVs by modifying their surface chemistry to include targeting peptides in an orientation-specific manner. Here, we describe a modular method to create targeted GVs using His-tagged antibody fragments, ensuring that the antibody fragments are connected to the GV in an orientation-specific manner. This is achieved via the functionalization of the GVs with nickel-nitrilotriacetic acid (Ni-NTA) group. First, we validated that these functionalized GVs can bind His-tagged green fluorescent protein and characterized the particle size and surface charge of functionalized GVs. Then, GVs targeted to prostate-specific membrane antigen (PSMA) using a minibody were validated using a knockout validation in vitro.

bioengineering↗

bioRxiv2025

Sono-uncaging for Spatiotemporal Control of Chemical Reactivity

Photo-uncaging - the use of light to reveal the active part of a chemical compound by photolysis of a protecting group - has long been used to study and actuate biochemical processes. However, light scattering limits the applications of photo-uncaging in opaque specimens or tissues. Here, we introduce sono-uncaging, a process in which a chemical functional group becomes exposed upon the application of ultrasound, which can be applied and focused in optically opaque materials. We engineered gas vesicles (GVs), air-filled protein nanostructures sensitive to ultrasound, to contain cysteines on their concealed inner surface, hypothesizing that the application of ultrasound would collapse the GV shell and reveal the cysteines. The resulting SonoCage construct reacted with monobromobimane (mBBr), a fluorogenic, thiol-reactive molecule, only after treatment with ultrasound, establishing the sono-uncaging proof of concept. We then demonstrated the spatial patterning capability of sono-uncaging by embedding the SonoCages in an mBBr-containing hydrogel and creating fluorescent patterns with phased array ultrasound. This patterning could be accomplished using a diagnostic imaging transducer. This work establishes sono-uncaging as a method for spatiotemporal control over chemical reactivity using widely available ultrasound technology.

bioengineering↗

bioRxiv2023

Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

synthetic biology↗