title: Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity.
abstract: Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Victor S
authors.initials: VS
authors.lastname: Lelyveld
authors.forename: Eric
authors.initials: E
authors.lastname: Brustad
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
authors.forename: Alan
authors.initials: A
authors.lastname: Jasanoff
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D003287:Contrast Media / Q000737:chemistry* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008279:Magnetic Resonance Imaging; D008345:Manganese / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011524:Protoporphyrins / Q000737:chemistry
chemical_list: D003287:Contrast Media; D008024:Ligands; D011524:Protoporphyrins; D008345:Manganese; D003577:Cytochrome P-450 Enzyme System; C028025:protoporphyrin IX