PROTEIN-BASED MOLECULAR SENSORS FOR HYPERPOLARIZED XENON NMR

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Degree type
Doctor of Philosophy (PhD)
Graduate group
Chemistry
Discipline
Chemistry
Biochemistry, Biophysics, and Structural Biology
Biochemistry, Biophysics, and Structural Biology
Subject
Contrast agent
Hyperpolarization
Molecular sensor
NMR
Protein engineering
Xenon
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Copyright date
2023
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Author
Zhao, Zhuangyu
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Abstract

To elucidate important biological processes, it is essential to employ molecular imaging techniques that provide excellent sensitivity, selectivity, and spatiotemporal resolution. Magnetic resonance imaging (MRI) is a non-invasive imaging technique with virtually unlimited penetration depth due to the radiofrequencies employed. However, conventional H-1 MRI techniques have intrinsic limitations, such as low sensitivity and lack of molecular details. To address these limitations, hyperpolarized (HP) Xe-129 NMR and MRI have been developed as complementary methods owing to the significant signal enhancement afforded by hyperpolarization and also xenon’s biocompatibility. HP Xe-129 in combination with chemical exchange saturation transfer (hyper-CEST) NMR improves the limit of detection to sub-picomolar range, in comparison to the hundreds of micromolar contrast agents typically required for H-1-based NMR detection. Furthermore, protein-based contrast agents offer many advantages over synthetic contrast agents, including molecular targeting specificity, genetic programmability, and low toxicity. Previously, our laboratory developed genetically encoded Xe-129 NMR contrast agents based on TEM-1 β-lactamase,maltose binding protein (MBP), and ribose binding protein (RBP). First, we employed molecular dynamics (MD) simulations and hyper-CEST NMR technique to elucidate the interaction of Xe with MBP. The Xe-129 hyper-CEST signal is highly programmable using an interdomain salt bridge K15-E111 in MBP. Next, we converted this salt bridge into a zinc binding site via rational design, which enabled sensitive and selective detection of cellular Zn(2+) by Xe-129 hyper-CEST. The same site can also be reconstructed using a s-tetrazine-based photocleavable linker, thus enabling optical control of magnetic resonance contrast. Finally, the split TEM-1 β-lactamase can be used to monitor protein-protein interactions in bacterial cells with NMR signal readouts. In conclusion, these results highlight the potential to develop proteins, especially the family of periplasmic binding proteins, as molecular sensors for NMR spectroscopy and MRI.

Advisor
Dmochowski, Ivan, J
Rhoades, Elizabeth
Date of degree
2023
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