Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine.
The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h's paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3-8.9 microM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity-dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.
Shapiro
Mikhail G
MG
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Westmeyer
Gil G
GG
Romero
Philip A
PA
Szablowski
Jerzy O
JO
Küster
Benedict
B
Shah
Ameer
A
Otey
Christopher R
CR
Langer
Robert
R
Arnold
Frances H
FH
Jasanoff
Alan
A
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D045744:Cell Line, Tumor; D003287:Contrast Media / Q000737:chemistry* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004298:Dopamine / Q000032:analysis* / Q000378:metabolism; D015195:Drug Design; D008279:Magnetic Resonance Imaging / Q000379:methods*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats
D001426:Bacterial Proteins; D003287:Contrast Media; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D004298:Dopamine