Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Dutka, Przemysław; Metskas, Lauren Ann; Hurt, Robert C.; Salahshoor, Hossein; Malounda, Dina; Lu, George J.; Chou, Tsui‐Fen; Shapiro, Mikhail G.; Jensen, Grant J.

doi:10.1016/j.str.2023.03.011

Cited by 27 publications

(38 citation statements)

References 76 publications

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“…We looked to introduce the motif into a region of GvpA that faces the GV exteriorthereby making it accessible to cytosolic FlAsHand which is tolerant of mutations to cysteine, such that the introduction of the TC tag does not abrogate GvpA expression and GV assembly. To predict which region of GvpA would best accommodate the TC tag, we looked to structural models of GvpA. , In a random mutagenesis experiment, the C-terminus of GvpA was found to be tolerant of many different point mutationsmore so than any other region of the proteinsuggesting that this region could be the most amenable to the substitution of the six-amino acid TC tag …”

Section: Results and Discussionmentioning

confidence: 99%

“…(b) Atomic model of two adjacent “ribs” composed of GvpA (PDB 8GBS). Interior- and exterior-facing sides of the GV shell are indicated. (c) Linear and atomic models of a single GvpA molecule with α helix and β-strand regions are indicated .…”

Section: Results and Discussionmentioning

confidence: 99%

“… Interior- and exterior-facing sides of the GV shell are indicated. (c) Linear and atomic models of a single GvpA molecule with α helix and β-strand regions are indicated . The C-terminal region of GvpA is shown as a dashed box in the linear representation of GvpA.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Gas vesicles (GVs) are air-filled protein nanostructures (∼85 nm diameter, ∼500 nm length) that are entering use in biomedical applications alongside other proteinaceous macromolecular assemblies such as encapsulins, virus-like particles, and nanocages. − In particular, GVs have recently emerged as promising agents for biomolecular ultrasound: they have been expressed recombinantly in both bacterial and mammalian cells and have been used as cavitation nuclei, ultrasonic reporters of cancer, acoustic actuators for selective cellular manipulation, and more. − These GV-based technologiesand those involving other macromolecular complexescould benefit from the knowledge of these structures’ subcellular localization, as this knowledge could enable the engineering of systems targeted to specific organelles or cellular compartments. However, there are currently no reported methods to fluorescently label GVs within cells.…”

Section: Introductionmentioning

confidence: 99%

“…To predict which region of GvpA would best accommodate the TC tag, we looked to structural models of GvpA. 1,16 In a random mutagenesis experiment, the C-terminus of GvpA was found to be tolerant of many different point mutations�more so than any other region of the protein�suggesting that this region could be the most amenable to the substitution of the six-amino acid TC tag. 1 Furthermore, the C-terminus of GvpA is on the exteriorfacing region of the protein 1 (Figure 1a−c).…”

Section: ■ Introductionmentioning

confidence: 99%

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Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures

Schrunk,

Dutka,

Hurt

et al. 2024

Bioconjugate Chem.

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Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' subcellular 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 reaction with the sixamino 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. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures

Schrunk,

Dutka,

Hurt

et al. 2024

Bioconjugate Chem.

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Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy

Ling,

Gungoren,

Yao

et al. 2024

Advanced Materials

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Nanotechnology offers significant advantages for medical imaging and therapy, including enhanced contrast and precision targeting. However, integrating these benefits into ultrasonography has been challenging due to the size and stability constraints of conventional bubble‐based agents. Here we describe bicones, truly tiny acoustic contrast agents based on gas vesicles, a unique class of air‐filled protein nanostructures naturally produced in buoyant microbes. We show that these sub‐80 nm particles can be effectively detected both in vitro and in vivo, infiltrate tumors via leaky vasculature, deliver potent mechanical effects through ultrasound‐induced inertial cavitation, and are easily engineered for molecular targeting, prolonged circulation time, and payload conjugation.This article is protected by copyright. All rights reserved

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50‐nm Gas‐Filled Protein Nanostructures to Enable the Access of Lymphatic Cells by Ultrasound Technologies

Shen,

Li,

Wang

et al. 2024

Advanced Materials

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Ultrasound imaging and ultrasound‐mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50‐nm gas‐filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as 50nmGVs. These diamond‐shaped nanostructures have hydrodynamic diameters smaller than commercially available 50‐nm gold nanoparticles and are, to our knowledge, the smallest stable, free‐floating bubbles made to date. 50nmGVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected 50nmGVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen‐presenting cells adjacent to lymphocytes. We anticipate that 50nmGVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas‐filled nanomaterials.This article is protected by copyright. All rights reserved

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Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Cited by 27 publications

References 76 publications

Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures

Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures

Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy

50‐nm Gas‐Filled Protein Nanostructures to Enable the Access of Lymphatic Cells by Ultrasound Technologies

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