Structure-guided directed evolution of highly selective P450-based magnetic resonance imaging sensors for dopamine and serotonin

Eric M. Brustad; Victor S. Lelyveld; Christopher D. Snow; Nathan Crook; Sang Taek Jung; Francisco M. Martinez; Timothy J. Scholl; Alan Jasanoff; Frances H. Arnold

doi:10.1016/j.jmb.2012.05.029

Structure-guided directed evolution of highly selective P450-based magnetic resonance imaging sensors for dopamine and serotonin

Eric M. Brustad, Victor S. Lelyveld, Christopher D. Snow, Nathan Crook, Sang Taek Jung, Francisco M. Martinez, Timothy J. Scholl, Alan Jasanoff, Frances H. Arnold

Research output: Contribution to journal › Article › peer-review

38 Citations (Scopus)

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.

Original language	English
Pages (from-to)	245-262
Number of pages	18
Journal	Journal of Molecular Biology
Volume	422
Issue number	2
DOIs	https://doi.org/10.1016/j.jmb.2012.05.029
Publication status	Published - 2012 Sept 14
Externally published	Yes

Bibliographical note

Funding Information:
We thank Fay Bi for technical assistance and P. Romero, M. F. Farrow, M. Smith, R. Lauchli, and P. Coehlo for helpful discussions during the preparation of the manuscript. We thank M. Shapiro for initiating work with BM3h-based contrast agents and for helpful discussions. We thank J. Kaiser, P. Nickolovski, J. Hoy, and the Molecular Observatory at the California Institute of Technology for assistance with high-throughput protein crystallography, X-ray data collection, and analysis. The Molecular Observatory is graciously supported by the Gordon and Betty Moore Foundation , the Beckman Institute , and the Sanofi-Aventis Bioengineering Research Program at Caltech . E.M.B. is supported by a Ruth M. Kirschstein National Institutes of Health (NIH) postdoctoral fellowship Award Number F32GM087102 from the National Institute of General Medical Sciences . We acknowledge support from the Jacobs Institute for Molecular Engineering for Medicine to F.H.A. This publication was made possible by grant 1R01DA028299‐01 from the NIH to A.J. and F.H.A. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.

Keywords

MRI contrast agents
biosensors
cytochrome P450
directed evolution
neurotransmitter

ASJC Scopus subject areas

Biophysics
Structural Biology
Molecular Biology

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10.1016/j.jmb.2012.05.029

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title = "Structure-guided directed evolution of highly selective P450-based magnetic resonance imaging sensors for dopamine and serotonin",

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.",

keywords = "MRI contrast agents, biosensors, cytochrome P450, directed evolution, neurotransmitter",

author = "Brustad, {Eric M.} and Lelyveld, {Victor S.} and Snow, {Christopher D.} and Nathan Crook and Jung, {Sang Taek} and Martinez, {Francisco M.} and Scholl, {Timothy J.} and Alan Jasanoff and Arnold, {Frances H.}",

note = "Funding Information: We thank Fay Bi for technical assistance and P. Romero, M. F. Farrow, M. Smith, R. Lauchli, and P. Coehlo for helpful discussions during the preparation of the manuscript. We thank M. Shapiro for initiating work with BM3h-based contrast agents and for helpful discussions. We thank J. Kaiser, P. Nickolovski, J. Hoy, and the Molecular Observatory at the California Institute of Technology for assistance with high-throughput protein crystallography, X-ray data collection, and analysis. The Molecular Observatory is graciously supported by the Gordon and Betty Moore Foundation , the Beckman Institute , and the Sanofi-Aventis Bioengineering Research Program at Caltech . E.M.B. is supported by a Ruth M. Kirschstein National Institutes of Health (NIH) postdoctoral fellowship Award Number F32GM087102 from the National Institute of General Medical Sciences . We acknowledge support from the Jacobs Institute for Molecular Engineering for Medicine to F.H.A. This publication was made possible by grant 1R01DA028299‐01 from the NIH to A.J. and F.H.A. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. ",

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doi = "10.1016/j.jmb.2012.05.029",

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AU - Crook, Nathan

AU - Jung, Sang Taek

AU - Martinez, Francisco M.

AU - Scholl, Timothy J.

AU - Jasanoff, Alan

AU - Arnold, Frances H.

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N2 - 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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Structure-guided directed evolution of highly selective P450-based magnetic resonance imaging sensors for dopamine and serotonin

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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