From Fluorescence to Magnetic Resonance: Engineering Proteins for Molecular Imaging

Research overview: My research interest broadly focuses on engineering proteins to develop new tools for  molecular imaging based on fluorescence and magnetic resonance imaging (MRI).

A. Genetically encoded fluorescent reporters for low-oxygen imaging

Low-O₂ regimes are encountered in several clinically and industrially relevant biosystems such as microbial fermentation, rumen microbiota, and biofilms. Hypoxic and anoxic regimes have hitherto pose a long-standing challenge to fluorescence imaging as widely used GFP-based probes depend on O₂ for fluorescence (Mukherjee et al., Curr. Op. Biotech., 2014). To address this challenge, I engineered bacterial & algal photoreceptors to develop viable fluorescent probes for live cell imaging & fluorimetry in low-O₂ environments. Specifically, I used directed evolution to enhance brightness (quantum yield) of a putative O₂-independent fluorescent reporter originally identified in Pseudomonas putida (Fig. 1) (Mukherjee et al., J. Biol. Eng., 2012). Second, I applied genome mining to identify and engineer algal photosensory proteins to develop two novel fluorescent reporters (Fig. 2) (Mukherjee et al., ACS Syn. Bio., 2014). Importantly, I demonstrated that, compared to GFP-based probes, one of the newly discovered algal fluorescent proteins (CreiLOV) is characterized by an overall small size (12 kDa), faster maturation kinetics, and improved performance at extremes of pH and temperature. Finally, I developed a comprehensive biophysical characterization framework to interrogate the maturation of fluorescence in this emerging class of O₂-independent reporter proteins with a view towards elucidating design rules for engineering improved reporters (Mukherjee et al., PLoS ONE., 2013). In this way, my research extends the spectrum of fluorescent imaging to systems that remain intractable using GFP-based reporters.

B. Genetically encoded reporters for magnetic resonance imaging

Despite the wide prevalence of MRI for clinical imaging, MRI remains a predominantly anatomical (vis-à-vis molecular) imaging modality due to a dearth of MR contrast agents that enable molecular-scale functional imaging. A highly promising albeit under-exploited approach towards addressing this challenge borrows from the GFP paradigm and involves the development of genetically targeted reporters for functional magnetic resonance imaging (Shapiro et al., 2010).  To this end, I recently engineered a Mn²⁺ binding bacterial enzyme (glutamine synthetase) to develop the first genetically encoded sensor for noninvasive detection of ATP using MRI (Fig. 3). As ATP fluctuations often accompany several neurodegenerative and cerebrovascular disorders, GS_ATP is a valuable tool to noninvasively and dynamically monitor ATP levels in cellular and animal models of stress, injury, inflammation, and toxicity.

C. Future research goals

Aim 1. Anaerobic imaging: I propose to leverage LOV-based fluorescent proteins to develop fluorescence complementation-dependent biosensors for imaging Ca²⁺, ATP, and cAMP dynamics in hypoxic models of cerebrovascular disease, tumor spheroids, and bacterial biofilms

Aim 2. Noninvasive MRI-based in vivo sensors:  In this aim, I seek to repurpose a eukaryotic antibacterial Mn²⁺ binding protein to develop a genetically encoded MRI-based sensor for calcium imaging in vivo. In addition, I propose to develop a new platform for noninvasive and dynamic imaging of protein interactions in preclinical animal models based on interaction-dependent reconstitution of split metalloproteins

Aim 3. Genetically encoded agents for photodynamic therapy:  I propose to engineer cyanobacterial light harvesting proteins as a first-of-its-kind class of genetically targetable therapeutic photosensitizers