a) Bioengineering of Gas Vesicles
Gas vesicles (GVs) are a class of hollow gas-filled protein nanostructures evolved in photosynthetic microbes. They are hundreds of nanometers in size and possess a gaseous interior enclosed by a ~ 2 nm-thick protein shell that is permeable to air but excludes water (Fig. a-b). The native role of GVs is to regulate the buoyancy of their microbial hosts in bodies of water so that when the microbes need to increase their photosynthesis rate, they will express GVs to float to the surface of the water for an increasing amount of sunlight. GVs are encoded by a cluster of 8-14 genes, and transferring these genes from their native hosts to other organisms, such as E. coli Nissle 1917 (the common probiotic Mutaflor®), results in the expression of GVs in these cells (Fig. c).
In 2014, GVs were engineered and repurposed as contrast agents for ultrasound imaging and hyperpolarized xenon MRI. These works leveraged the unique physical properties of these protein nanostructures: the change of acoustic impedance at the air-water interface enables ultrasound imaging, and the free diffusion of xenon molecules across GV shells permits HyperCEST MRI (see references below). In 2018, E. coli and and Salmonella typhimurium were genetically engineered to express a highly echogenic genotype of GVs, which rendered these cells to be imaged non-invasively within the gastrointestinal tract and in tumors (Fig. d). This offers a potential route for studying the microbiome and monitoring cancer progression and therapy. In this nascent field, the biodiversity, biochemistry and physical properties of GVs provide rich opportunities for their utilities in many directions for imaging and control of cellular functions.
b) Biomolecular Contrast Agents for MRI
Magnetic resonance imaging (MRI) is a widely established medical imaging modality that can observe functions of specific cells and molecules inside living organisms. Additionally, MRI utilizes magnetic field and radiofrequency waves that can penetrate through biological tissues without attenuation, which is in contrast to ~ 1 mm penetration depth of light for optical imaging. However, compared to optical imaging, in which fluorescent protein reporters are available to visualize cellular functions with high specificities, such as gene expression and neural activation, to date relatively few such reporters are available for MRI. To enable the use of MRI to probe molecular events in deep-lying tissue with high molecular and cellular specificity, we aim to engineer special proteins that can interact with the magnetic field and is thus visible in MRI images. As an example, the protein engineering of an iron-containing P450 enzyme enables the signal intensity of T1-weighted MRI images to correlate specifically to the concentration of dopamine in brains of living rats (see Figure).
c) Solid-state NMR Spectroscopy on Membrane Protein
Nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and cryo-electron microscopy are three main methods that can determine atomic-resolution protein structures. Unlike the other two, NMR is unique in its ability to study proteins in their native environment of pH, hydration and temperature, or even directly proteins in living cells. This is beneficial in scenarios where the cellular context is crucial for the correct folding, dynamics and function of the proteins.
NMR method can be subdivided into the more established solution NMR and solid-state NMR (ssNMR). ssNMR is not restricted by the requirement of fast isotropic tumbling of proteins in a solution. As an example, ssNMR can study membrane proteins reconstituted in native lipid bilayers, where the whole protein-lipid assembly does not undergo isotropic motion. Thus, ssNMR may provide atomic-resolution structure, dynamics and interaction information for membrane proteins, which compared to soluble proteins, are less understood due to the technical challenges to studying them by most of the structural biology methods.