Directed Evolution of Protein-Based Neurotransmitter Sensors for MRI

  • Philip A. Romero
  • Mikhail G. Shapiro
  • Frances H. Arnold
  • Alan Jasanoff
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 995)

Abstract

The production of contrast agents sensitive to neuronal signaling events is a rate-limiting step in the development of molecular-level functional magnetic resonance imaging (molecular fMRI) approaches for studying the brain. High-throughput generation and evaluation of potential probes are possible using techniques for macromolecular engineering of protein-based contrast agents. In an initial exploration of this strategy, we used the method of directed evolution to identify mutants of a bacterial heme protein that allowed detection of the neurotransmitter dopamine in vitro and in living animals. The directed evolution method involves successive cycles of mutagenesis and screening that could be generalized to produce contrast agents sensitive to a variety of molecular targets in the nervous system.

Key words

Magnetic resonance imaging Directed evolution Protein engineering Cytochrome P450 Dopamine 

References

  1. 1.
    Jasanoff A (2007) MRI contrast agents for functional molecular imaging of brain activity. Curr Opin Neurobiol 17:593–600PubMedCrossRefGoogle Scholar
  2. 2.
    Gilad AA et al (2007) Artificial reporter gene providing MRI contrast based on proton exchange. Nat Biotechnol 25:217–219PubMedCrossRefGoogle Scholar
  3. 3.
    McMahon MT et al (2008) New “multicolor” polypeptide diamagnetic chemical exchange saturation transfer (DIACEST) contrast agents for MRI. Magn Reson Med 60:803–812PubMedCrossRefGoogle Scholar
  4. 4.
    Shapiro MG, Szablowski JO, Langer R, Jasanoff A (2009) Protein nanoparticles engineered to sense kinase activity in MRI. J Am Chem Soc 131:2484–2486PubMedCrossRefGoogle Scholar
  5. 5.
    Iordanova B, Robison CS, Ahrens ET (2010) Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate. J Biol Inorg Chem 15:957–965PubMedCrossRefGoogle Scholar
  6. 6.
    Shapiro MG et al (2010) Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine. Nat Biotechnol 28:264–270PubMedCrossRefGoogle Scholar
  7. 7.
    Dougherty MJ, Arnold FH (2009) Directed evolution: new parts and optimized function. Curr Opin Biotechnol 20:486–491PubMedCrossRefGoogle Scholar
  8. 8.
    Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378PubMedGoogle Scholar
  9. 9.
    Bernstein MA, King KF, Zhou XJ (2004) Handbook of MRI pulse sequences. Academic, New YorkGoogle Scholar
  10. 10.
    Fasan R, Chen MM, Crook NC, Arnold FH (2007) Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties. Angew Chem Int Ed Engl 46:8414–8418PubMedCrossRefGoogle Scholar
  11. 11.
    Bloom JD, Labthavikul ST, Otey CR, Arnold FH (2006) Protein stability promotes evolvability. Proc Natl Acad Sci U S A 103:5869–5874PubMedCrossRefGoogle Scholar
  12. 12.
    Lewis DFV (2001) Guide to cytochrome P450 structure and function. Taylor & Francis, New YorkCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Philip A. Romero
    • 1
  • Mikhail G. Shapiro
    • 2
  • Frances H. Arnold
    • 1
  • Alan Jasanoff
    • 2
    • 3
    • 4
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA
  2. 2.Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Department of Brain and Cognitive SciencesMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Department of Nuclear Science and EngineeringMassachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations