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: 3.91). 05/2012; 422(2):245-62. DOI: 10.1016/j.jmb.2012.05.029
Source: PubMed

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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: A major challenge in neuroscience is to decipher the logic of neural circuitry and to link it to learning, memory, and behavior. Synaptic transmission is a critical event underlying information processing within neural circuitry. In the extracellular space, the concentrations and distributions of excitatory, inhibitory, and modulatory neurotransmitters impact signal integration, which in turn shapes and refines the function of neural networks. Thus, the determination of the spatiotemporal relationships between these chemical signals with synaptic resolution in the intact brain is essential to decipher the codes for transferring information across circuitry and systems. Here, we review approaches and probes that have been employed to determine the spatial and temporal extent of neurotransmitter dynamics in the brain. We specifically focus on the design, screening, characterization, and application of genetically encoded indicators directly probing glutamate, the most abundant excitatory neurotransmitter. These indicators provide synaptic resolution of glutamate dynamics with cell-type specificity. We also discuss strategies for developing a suite of genetically encoded probes for a variety of neurotransmitters and neuromodulators.
    ACS Chemical Neuroscience 01/2015; 6(1). DOI:10.1021/cn500280k · 4.21 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Heme proteins are among the most abundant and important metalloproteins, exerting diverse biological functions including oxygen transport, small molecule sensing, selective CH bond activation, nitrite reduction, and electron transfer. Rational heme protein designs focus on the modification of the heme-binding active site and the heme group, protein hybridization and domain swapping, and de novo design. These strategies not only provide us with unique advantages for illustrating the structure-property-reactivity-function (SPRF) relationship of heme proteins in nature but also endow us with the ability to create novel biocatalysts and biosensors.
    Chemistry - An Asian Journal 05/2013; 8(11). DOI:10.1002/asia.201300291 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We demonstrate a technique for mapping brain activity that combines molecular specificity and spatial coverage using a neurotransmitter sensor detectable by magnetic resonance imaging (MRI). This molecular functional MRI (fMRI) method yielded time-resolved volumetric measurements of dopamine release evoked by reward-related lateral hypothalamic brain stimulation of rats injected with the neurotransmitter sensor. Peak dopamine concentrations and release rates were observed in the anterior nucleus accumbens core. Substantial dopamine transients were also present in more caudal areas. Dopamine-release amplitudes correlated with the rostrocaudal stimulation coordinate, suggesting participation of hypothalamic circuitry in modulating dopamine responses. This work provides a foundation for development and application of quantitative molecular fMRI techniques targeted toward numerous components of neural physiology.
    Science 05/2014; 344(6183):533-535. DOI:10.1126/science.1249380 · 31.48 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014