(6ff) 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