[show abstract][hide abstract] ABSTRACT: 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.
Journal of Molecular Biology 05/2012; 422(2):245-62. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: A poly(para-phenylene ethynylene) with water-solubilizing groups and Gd(III) chelates conjugated to the polymer backbone was designed and synthesized. Pre- and post-polymerization functionalization approaches were explored and the pre-polymerization approach for the introduction of the Gd(III) chelate was found to be more successful. The UV–vis absorption and fluorescence emission properties of the protected polymers were characterized and were found to be consistent with the results expected for this class of polymers. Removal of the protecting groups followed by chelation of Gd(III) led to a water-dispersible polymer. Relaxivity measurements were performed on this polymer with the aim of evaluating its potential as a new MRI contrast agent, and an r1 of 1.37 L mmol–1 s–1 at 310 K and 20 MHz was determined. These results, along with dynamic light scattering analyses, suggested that the polymers formed micrometre-sized assemblies in aqueous solution. Although the relaxivity was relatively modest, these results provide important insights into the assembly properties of this new class of polymers and into the design criteria for future agents.
Canadian Journal of Chemistry 12/2010; 89(1):47-56. · 0.96 Impact Factor