Structure-Guided Directed Evolution of Highly Selective P450-Based Magnetic Resonance Imaging Sensors for Dopamine and Serotonin

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 05/2012; 422(2):245-62. DOI: 10.1016/j.jmb.2012.05.029
Source: PubMed


New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450-BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high-affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450-BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins.

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Available from: Timothy James Scholl, Dec 30, 2013
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    • "(A) Protein-based T 1 contrast agents operate largely through an inner sphere relaxation mechanism dependent on metal-coordinated water molecules in fast exchange with bulk solvent. In the example shown (top), the heme iron (brown) of an engineered P450 BM3 variant (gray ribbon structure) interacts with an axial inner sphere water ligand (blue ball, arrowhead) [24]. Bound water protons (not shown) experience dipole coupling with the metal ion and undergo relaxation. "
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